BENZO(a)PYRENE
First draft prepared by Dr J.C. Larsen,
Institute of Toxicology, National Food Agency of Denmark.
1. EXPLANATION
Benzo(a)pyrene (B(a)P) is a contaminant that occurs
ubiquitously in the environment together with other polycyclic
aromatic hydrocarbons (PAHs) as a product of incomplete combustion
or pyrolysis of organic material containing carbon and hydrogen. In
addition to natural sources of PAHs (e.g., forest fires), there are
numerous man-related combustion processes which result in
contamination of air, water, food, soil and sediment. Main sources
of B(a)P and also PAHs in the environment are residential heating
(coal and wood-burning stoves and fireplaces), industrial plants
(refuse burning, smelting, coke production), vehicle exhausts and
cigarette smoke. (Fazio & Howard, 1983; Grimmer, 1979; Bjorseth,
1983.).
Humans may be exposed to PAHs from air, water, food and tobacco
smoke. While the majority of studies have concentrated on the
determination of B(a)P, it is important to note that it constitutes
1 to 20% of estimated total carcinogenic PAHs present and usually
less than 5% of total PAHs (Fazio & Howard, 1983); Bjorseth, 1983).
B(a)P may contaminate foods via deposition of airborne
particulate matter, by direct drying with smoke, or by absorption
during the smoking process. Also high temperature heat-processing
of food may lead to contamination with B(a)P and other PAHs.
Benzo(a)pyrene has not previously been evaluated by the Joint
FAO/WHO Expert Committee on Food Additives.
B(a)P has been evaluated for carcinogenicity by IARC
(International Agency for Research on Cancer) (IARC, 1973; 1982;
1983). The following evaluations were made for B(a)P: Inadequate
evidence for carcinogenicity to humans; sufficient evidence for
carcinogenicity to animals; sufficient evidence for activity in
short-term tests (IARC, 1982; 1983).
1.1 Dietary exposure
Sources of B(a)P contamination of food-stuffs are numerous,
varied, and widespread. They include contaminated air, water, soil
and sediment, modes of cooking (i.e., charcoal grilling), food
processing (e.g., smoke curing, flue-drying), and food additives
(e.g., smoke flavorings).
There are two major sources of the occurrence of B(a)P in
foods. The most important source is probably the deposition and
uptake of B(a)P and other PAHs from polluted air on food crops.
This makes cereals, vegetables, fruits and vegetable oils important
contributors to the intake of B(a)P by humans. In particular,
drying of cereals and plants used for production of crude vegetable
oils using direct application of combustion gases can result in
contamination of the product with PAHs. Kale, lettuce, barley, rye,
and wheat are examples of crops that can be contaminated with B(a)P
from air pollution. In kale 4.2 to 15.6 µg B(a)P/kg has been found
in various locations in Western Germany, and wheat samples from
rural areas contained 0.19 to 0.34 µg B(a)P/kg, whereas 0.72 to 3.52
µg B(a)P/kg was measured in wheat grown near industrial plants. In
crude vegetable oils 1.2 to 15.3 µg B(a)P/kg has been reported. The
content of B(a)P in fruit depends on the site of growth. In
different sorts of fruit 0.2 to 0.5 µg B(a)P/kg was found in
residential areas, while 30 to 60 µg B(a)P/kg was found near
industrial plants (Grimmer & Pott, 1983). Certain seafoods,
especially filter feeders (e.g. clams, oysters) normally have higher
levels than do finfish (Vaessen et al., 1984).
The other significant source is the formation and deposition of
PAHs during heat processing using methods such as roasting, smoking,
and grilling. Lijinsky and Shubik (1964) identified B(a)P and other
PAHs in charcoal-broiled meat at an average level of 8 µg B(a)P/kg
steak. It is important to note that the formation of PAHs is only
significant at higher temperatures, generally over 350-400° C, and
that below this temperature the endogenous formation of B(a)P in the
food is minimal. Thus, cooking procedures using heat conduction,
such as pan-frying, or radiation, as in electric broiling and
baking, do not lead to significant formation of B(a)P in food (Toth
& Potthast, 1984). When meat was placed directly in contact with
the flames of a log fire a significant amount of B(a)P was formed
(6-212 µg B(a)P/kg meat) (Larsson et al., 1983). The B(a)P
content of grilled food primarily stems from the fuel used and from
the pyrolysis of fat dripping down on the heat source. Among the
fuels normally used in grilling, charcoal yielded only small amounts
of PAHs (0.1-1.0 µg B(a)P/kg food), while smoldered spruce or pine
cones yielded 2 to 31 µg B(a)P/kg meat (Larsson et al., 1983).
The fat content of the meat is also important. The more fat that
drips on the fuel the more PAHs may be formed and deposited on the
meat (Lijinsky & Ross, 1967; Toth & Blaas, 1973; Doremire et al.,
1979). Increasing the fat content of charcoal-grilled ground beef
patties from 15% to 40% increased the B(a)P content from 16 to 121
µg/kg (Fretheim, 1983).
Smoking of food may be another source of B(a)P. Curing smoke is
normally produced from wood (sawdust). In traditional smoking the
smoke is generated at the bottom of the oven and the food is placed
directly over the smoking wood. In modern industrial smoking ovens
the smoke is generated in a separate chamber and led into the
smoking chamber where the products are placed. This gives a better
control of the smoking process. In various investigations the
average values ranged from 0.2 to 0.9 µg B(a)P/kg product for smoked
meat products, such as sausages, ham, bacon, etc. Similar B(a)P
concentrations have been found in the edible part of smoked fish.
Larsson (1982) found 0.4 to 2.7 µg B(a)P/kg in a survey of Swedish
smoked fish. Very high values (23 to 55 µg/kg) may be found in
intensively smoked products (black-smoked) (Grimmer & Pott, 1983).
Various estimates of the dietary intake of B(a)P have been
made. Based on an average life expectancy of 70 years an inhabitant
of East Germany was estimated to ingest a total of 24 to 85 mg of
B(a)P. This corresponds to approximately 1.0 to 3.3 µg/day. The
major part of B(a)P was estimated to be ingested from cereals and
vegetable oils (Fritz, 1971). Santodonato et al., (1980; 1981)
suggested on the basis of a total daily food consumption by man from
all types of foods of 1600 g/day and an estimated typical range of
concentrations for B(a)P of 0.1-1.0 ppb in foods that the possible
dietary intake of B(a)P was 0.16-1.6 µg/day. In comparison, the
intakes of B(a)P from air, water, and cigarette smoking were
estimated at 0.0095-0.0435, 0.0011, and 0.44 µg/day, respectively.
In a UK survey the average intake of B(a)P was estimated at 0.25 µg
B(a)P/person/day (total PAHs/intake: 3.7 µg/person/day). Cereals
and oils/fats accounted for 80% of the intake (Dennis et al.,
1983). Very similar figures were estimated in a Swedish survey on
the intake of PAHs with the diet (Larsson, 1986).
Human exposure to B(a)P from inhalation and food was compared
for 10 homes in Phillipsburg, New Jersey, a city that contains a
metal pipe foundry, which is a suspected major source of B(a)P. The
mean outdoor concentration of B(a)P was 0.9 ng/m3, and the indoor
concentrations ranged from 0.1-8.1 ng/m3. Food samples were
acquired from family meals each day. The range of B(a)P per gram of
wet weight of food was between 0.004 and 1.2 ng/g. Of the 20 weeks
of exposure (10 x 2 weeks), 10 had higher food exposures and the
other 10 had higher inhalation exposures. Of the two groups, the
higher food exposures usually had a greater number of ng of
B(a)P/week. The weekly ingestion of B(a)P with food ranged from 10
to 4000 ng (Lioy et al., 1988).
In a "human diet", used for animal experiments, and prepared
according to mean levels of consumption in The Netherlands, and
containing fried meat, baked bread, cereals, fruits, and vegetables,
the B(a)P content was determined to be 0.15 µg/kg (Alink et al.,
1989).
2. BIOLOGICAL DATA
The toxicological literature on B(a)P is overwhelming.
However, the majority of toxicological studies on B(a)P have
addressed the question of B(a)P as an air pollutant, potentially
implicated in lung cancer in man, and therefore have not used oral
application. B(a)P has been the model compound per se in chemical
carcinogenesis, and has been intensively tested for carcinogenicity
in model systems such as the skin carcinogenesis models, for
interactions with DNA in vitro and in vivo, for in vitro
activation in numerous cell types, and has been used as a positive
control in a multitude of short-term in vitro and in vivo test
systems. This monograph is centered on studies using oral
application of B(a)P, while studies using other routes are primarily
included when they add to an understanding of B(a)P toxicology and
are deemed valuable for the evaluation of B(a)P as a contaminant in
food.
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
The absorption, distribution and excretion of B(a)P have been
reviewed by IARC (1983).
After subcutaneous and intravenous administrations of B(a)P to
rats the main excretory route was the bile. About 1% of the dose
was recovered as unchanged B(a)P in the faeces (Chalmers & Kirby,
1940).
When 14C-B(a)P (0.45 mg) was given intravenously to female
rats a rapid uptake by the liver and subsequent elimination in the
bile were seen. In 24 hours 65% of the radioactivity had been
excreted in the faeces and 18% in the urine, while 1.8% remained in
the liver (Heidelberger & Weiss, 1951).
The pattern of distribution of 14C-B(a)P was similar after
subcutaneous, intravenous, and intratracheal administration to
strain A mice and Wistar rats. After intravenous administration (11
µg) radioactivity rapidly disappeared from the blood and peak levels
were obtained in the liver. Minimal localization was seen in
spleen, kidney, lung, and stomach. After 24 hours 50-60% of the
radioactivity had been recovered from intestine and faeces and 8-13%
from urine (Kotin et al., 1959).
Female Sprague-Dawley rats were given intravenously 10 µg of
14C-B(a)P. After 15 minutes 7% of the dose had been excreted in
the bile and after 600 minutes the cumulative excretion amounted to
53% of the radioactivity. Pretreatment of the rats with 20 mg
B(a)P/kg body weight for 7 days enhanced the initial rate of biliary
excretion of 14C-B(a)P but not the total excretion. No
accumulation of B(a)P was seen in body fat (Schlede et al., 1970a;
1970b).
Bock and Dao (1961) showed relatively high localization of
B(a)P in the mammary gland and general body fats after a single
feeding of 10-30 mg B(a)P to rats.
B(a)P (50-150 mg/kg body weight) was readily absorbed from the
gastrointestinal tract of the female Sprague-Dawley rat and the
concentration of B(a)P in adipose and mammary tissues increased
exponentially with the dose. In another experiment peak levels of
B(a)P were found in the cannulated thoracic lymph duct 3-4 hours
after treatment. Ten to 20% of the dose was recovered in the lymph
(Rees et al., 1971).
Radioactivity was excreted in the milk from lactating rabbits
and sheep when 1 mg of 14C-B(a)P was given in their diet. The
amount excreted in 6 days was 0.003% of the dose in rabbits, and
0.01% of the dose in the sheep (West & Horton, 1976).
One hour after intravenous administration of 14C-B(a)P to
lactating rats the average amount of radioactivity detected was
0.21% of the administered dose per ml of milk as compared with 0.17%
per ml of blood (LaVoie et al., 1987).
A large fraction (45%) of unchanged B(a)P in rat blood was
associated with serum lipoproteins while only 8% of the metabolites
was associated with this component. Forty to forty-five percent of
each was associated with red blood cells. Clearance of B(a)P by the
perfused rat liver greatly depended on the presence of serum
lipoproteins and albumin in the medium perfusing the organ (Wiersma
et al., 1984).
Male Sprague-Dawley rats with biliary and mesenteric lymphatic
catheters received intraduodenally a dose of 0.4 µmol (100 µg) 3H-
B(a)P in different amounts of olive oil. Cumulative radioactivity
recovered in 24 hours was 20% of the dose, regardless of the amount
of oil used as vehicle. Eighty percent of the material was found in
bile. It was concluded that the lymphatics play a limited role in
the systemic entry of orally administered B(a)P (Laher et al.,
1984).
Conscious rats with bile duct and duodenal catheters were given
1 mg of 3H-B(a)P intraduodenally in corn oil with or without
exogenous bile. Cumulative recovery of radiolabel in bile and urine
over 24 hours showed that in the presence of bile 30% of the
radiolabel was recovered. In the absence of bile only 7% was found
(Rahman et al., 1986).
B(a)P was found distributed to all regions of male Wistar rat
brain after an intraperitoneal injection of 15 nmole. The highest
level was found in liver, followed by kidney, lung, brain (Das et
al., 1985a).
3H-B(a)P was administered by intratracheal instillation (1
µg/kg body weight) to male Sprague-Dawley rats, and the amount of
radioactivity in various organs was determined at timed intervals
between 5 and 360 min. Radioactivity in liver increased rapidly,
reaching a maximum of 21% of the dose within 10 minutes after
instillation. The carcass accounted for 15-30% of the dose within
the time intervals investigated. B(a)P disposition indicated that
enterohepatic circulation of metabolites was occurring (Weyand &
Bevan, 1986).
Intestinal absorption, bioavailability, hepatic and pulmonary
extraction, and elimination of low doses of 3H-B(a)P (0.7-4.4 nmol)
were studied in the male Sprague-Dawley rat. The hepatic extraction
ratio was 0.4 both in a liver perfusion model and in vivo as
determined by comparison of intravenous and intraportal infusion
experiments in anaesthetized rats. The pulmonary extraction ratio
in vivo was 0.11. Analysis of B(a)P concentrations in atrial
blood and in the bile after continuous B(a)P infusion into the
duodenum of anaesthetized rats indicated that at least 30% of the
dose was absorbed from the gut. When B(a)P was given by gavage
about 10% of the dose escaped the liver and appeared in the blood
(Foth et al., 1988).
2.1.2 Biotransformation
The biotransformation of B(a)P and other polycyclic aromatic
hydrocarbons has been reviewed extensively (Sims & Grover, 1974;
Gelboin, 1980; Levin et al., 1982; Pelkonen & Nebert, 1982;
Conney, 1982; IARC, 1983; Cooper et al., 1983; Grover, 1986).
B(a)P is initially oxidized, primarily by the microsomal NADPH-
dependent cytochrome P-450 monooxygenase system, to several arene
oxides. The arene oxides may rearrange spontaneously to phenols (3-
OH-, 7-OH-, 9-OH-, and 6-OH-B(a)P), undergo hydration to the
corresponding trans-dihydrodiols (catalyzed by microsomal epoxide
hydrolase), or may react covalently with glutathione, either
spontaneously or catalyzed by cytosolic glutathione-S-transferases.
6-OH-B(a)P is further oxidized to the 1,6-, 3,6-, or 6,12-quinones;
3-OH-B(a)P can be oxidized to the 3,6-quinone, and 9-OH-B(a)P can be
oxidized to the 4,5-oxide, which is hydrated to the corresponding
4,5-dihydrodiol. The phenols, quinones and dihydrodiols can all be
conjugated to glucuronides and sulfate esters, and the quinones also
form glutathione conjugates (IARC, 1983).
The dihydrodiols may undergo further oxidative metabolism. A
number of unidentified metabolites are formed from the 4,5-
dihydrodiol, and the 9,10-dihydrodiol can be oxidized to its 1-
and/or 3-phenol derivatives. The principal route of oxidative
metabolism of the B(a)P-7,8-dihydrodiol is to the B(a)P-7,8-
dihydrodiol-9,10 epoxide, which has been implicated as the most
important reactive metabolite of B(a)P for its mutagenic and
carcinogenic properties. The diol-epoxides can be conjugated with
glutathione or hydrolyse spontaneously to tetrols. Thus, B(a)P may
undergo a number of complex simultaneous and sequential
biotransformations. The situation is even more complex as the diol-
epoxides each may exist in 4 optically active isomers (each
diastereoisomer can be resolved into two enantiomers). In rat liver
microsomes the (+)-[7R,8S]-oxide of B(a)P is formed in a 20-fold
excess relative to the (-)-[7S,8R]-oxide and is stereospecifically
metabolized by epoxide hydrolase to the (-)-[7R,8R]-dihydrodiol
(Thakker et al., 1977), which in turn is further oxidized to (+)-
B(a)P-7,8-diol-9,10-epoxide-2[7R,8S,9S,10R] (variously called: diol-
epoxide-2; B(a)PDEI; trans-B(a)PDEI; (+)-anti-B(a)PDE). B(a)PDEI is
the predominant diol-epoxide formed from B(a)P-7,8-dihydrodiol in
almost all tissues examined, and is also the only isomer with high
tumorigenic activity. It is the predominant isomer found covalently
bound to DNA (forming the N2-10ß -[7ß, 8alpha, 9alpha-trihydroxy-
7,8,9,10-tetrahydrol B(a)P]-yl)deoxyguanosine adduct; B(a)PDEI-dGuo)
in a variety of mammalian cells and organs exposed to B(a)P.
Another diol epoxide B(a)PDEI ((-)syn-B(a)PDE; cis-B(a)PDEII;
(-)diol-epoxide 1) is also formed in significant amounts, while the
two other diol-epoxides are formed in only very small amounts. The
formation of diol-epoxides can also be catalyzed by microsomal
prostaglandin synthetase, present in a variety of different tissues
(Pezzuto et al., 1978; Levin et al., 1982; Conney, 1982; IARC,
1983; Cooper et al., 1983).
2.1.2.1. In vitro
An overview of the in vitro metabolism of B(a)P by various
human tissues was given by IARC (1983).
Primary hepatocyte cultures from six human donors metabolized
B(a)P to a significant extent (24-35 nmol in 24 hours). The
predominant extracellular organic solvent-soluble B(a)P metabolites
were the 9, 10- and 7, 8-dihydrodiols, 9-hydroxy-B(a)P, and a
mixture of tetrols, but the general ratios of these metabolites
varied widely among the cells from different donors (Moore & Gould,
1984).
Monolayer cultures of human bronchial epithelial cells
converted B(a)P to dihydrodiols, phenols, quinone derivatives, and
polyhydroxylated forms. Sulfate and glucuronide conjugates of B(a)P
metabolites were also detected. Both total metabolism and
distribution of metabolites showed a 10-fold or greater variation in
cultures from different specimens. When the data were divided
according to smoking status, however, no differences in total
metabolism, extent of conjugation, or distribution of metabolites
could be demonstrated between the two groups (Siegfried et al.,
1986).
The capacity of human hepatocytes to metabolize B(a)P was not
saturated at up to 100 µM of B(a)P, and the predominant metabolites
produced were a mixture of highly polar B(a)P forms. The next four
most prevalent forms of B(a)P metabolites were the 3-hydroxy B(a)P,
B(a)P-4,5-dihydrodiol, B(a)P-9,10-dihydrodiol, and B(a)P-7,8-
dihydrodiol. These metabolites all increased nearly linearly with
dose. B(a)P metabolite binding to DNA was associated with the
amount of unconjugated B(a)P-7,8-dihydrodiol metabolite (Monteith
et al., 1987).
In human liver microsomes a four-fold variation in B(a)P
metabolism was observed. The levels of expression of cytochromes P-
450 from five gene subfamilies did not show any correlation with the
rate of B(a)P metabolism. The P450IA1 was most effective in
metabolizing B(a)P, but several other forms of cytochrome P-450 were
shown to be involved in B(a)P metabolism in humans (Hall et al.,
1989).
The ability of seven different forms of cytochrome P-450
purified from rat liver microsomes to metabolize B(a)P was compared.
The major 3-methylcholanthrene (MC) inducible cytochrome P-450 (form
c) exhibits the greatest activity toward B(a)P. Cytochrome P-450d,
a minor MC-inducible from, has far lower activity for metabolism of
B(a)P. Two phenobarbital (PB)-induced forms (P-450's b and e) had
low activity. P-450's a, h, and pregnenolone-16 alpha-carbonitrile
(PCN) exhibited little activity toward B(a)P (Wilson et al.,
1984).
3-OH-B(a)P and the 1,6- and 3,6-quinones were the major
products formed by a reconstituted pulmonary cytochrome P-450MC
system. The B(a)P-9,10-dihydrodiol was the major dihydrodiol formed
by pulmonary cytochrome P-450MC. The addition of purified epoxide
hydrolase increased the formation of B(a)P-dihydrodiols,
particularly B(a)P-7,8-dihydrodiol. Similar results were obtained
in reconstituted systems of hepatic cytochrome P-450MC (Sagami et
al., 1987).
When incubated in the presence of peroxidising polyunsaturated
fatty acids such as linoleic acid (C18:2), arachidonic acid (C20:4),
eicosapentanoic acid (C20:5) or docosahexanoic acid (C22:6) B(a)P
was converted to oxidized products. Between 7% and 9% of the B(a)P
was oxidized in one hour when incubated with arachidonic acid and
docosahexanoic acid. 1,6-,3,6-, and 6,12-quinone derivatives of
B(a)P were identified by HPLC. The products of B(a)P oxidation were
shown to produce sister chromatid exchange (SCE) in CHV79 cells
(McNeill & Willis, 1985).
Homogenates of colonic mucosa from different mouse strains
metabolized B(a)P to predominantly phenolic derivatives (3-OH- and
9-OH-B(a)P), and lesser amounts of diols (4,5-, 7,8-, and 9,10-
B(a)P-diol) and quinones (1,6-, 3,6-, and 6,12-B(a)P-quinone)
(Anderson et al., 1982).
When male C57BL/6 mice were fed a basal semisynthetic diet with
added various amounts of vitamins and/or types of fibers and/or
types of fats no differences were observed in the ability of colonal
mucosal homogenates to metabolize B(a)P. The various diets had no
effects on beta-naphthoflavone induced metabolism (Anderson et al.,
1987).
In mouse liver microsomes the 7,8-epoxidation of B(a)P, and the
9, 10-epoxidation of B(a)P trans-7,8-dihydrodiol coupled with
covalent binding of the highly reactive diol-epoxide, were shown to
be mediated by P-450 protein(s) that are responsible for aryl
hydrocarbon hydroxylase activity and that are coordinately
controlled by the Ahb allele (Van Canfort et al., 1985).
B(a)P metabolites formed by rough and smooth endoplasmic
reticulum, nuclei, and plasma membrane as well as mitochondrial
fractions were investigated. The metabolic profiles produced by the
two most active fractions, smooth and rough endoplasmic reticulum,
were very similar to each other but different from those produced by
the other three preparations. The metabolite pattern produced by
incubations containing nuclear fractions differed slightly from that
produced by the fractions of endoplasmic reticulum, but plasma
membrane and mitochondria produced markedly different patterns
(Oesch et al., 1985).
When groups of 6-8 male Wistar rats were fed diets containing
10% of different types of fat, there were significant changes in the
incorporation of fatty acids into the endoplasmic reticulum of the
mucosal cells of the small intestine: the proportions of
polyunsaturated fatty acids in the endoplasmic reticulum reflected
the amounts of these fatty acids in the dietary fat. The rate of
B(a)P in vitro oxidation in the intestinal mucosa was dependent on
the amount and composition of the dietary fat, but the range and
proportions of the metabolites produced were not effected. Dietary
C18:2 (corn oil) was particularly important in elevating the rate of
B(a)P oxidation, but dietary C20:5 and C22:6 (mackerel oil and cod
liver oil) also effectively increased the rate of B(a)P oxidation
(Gower & Wills, 1986).
Levels of total metabolism of B(a)P by rat and human kidney
cells were similar, although analysis of specific metabolites of
B(a)P indicated that species differences existed. Human kidney
cells produced the organic-soluble metabolites B(a)P-9,10-diol,
B(a)P-4,5-diol, B(a)P-7,8-diol, B(a)P-3,6-quinone, and B(a)P-9-
phenol. Rat kidney cells produced organic-soluble B(a)P-pre-9,10-
diols, B(a)P-9,10-diol, B(a)P-4,5-diol, and B(a)P-6,12-quinone.
Both species produced sulfate and glucuronide conjugates of all
products (Rudo et al., 1989).
The metabolism of 1 mM benzo(a)pyrene was studied in isolated
perfused lung and liver of 5,6-benzoflavone-pretreated rats.
Benzo(a)pyrene metabolism by the liver was more rapid than by the
lung, but total metabolite formation in the lung at the end of a
120-min perfusion period was comparable to that in the liver. Lung
perfusate was characterized by high concentrations of free
metabolites, diols outweighing phenols; in liver perfusate free
metabolite concentrations were low, and large quantities of
metabolites were found as conjugates in the bile at the end of
perfusion. The tissue concentrations of free diols and phenols
including the precursors of the main DNA-binding secondary
metabolites were higher in the lung than in the liver (Molliere et
al., 1987).
The oesophagi of anaesthetized hamsters were surgically
catheterized so that radiolabeled material instilled as B(a)P in the
nose could be collected and analyzed for metabolites. About 50% of
the instilled B(a)P was metabolized in the nose and, potentially,
would have been swallowed in an awake animal. Using homogenates of
respiratory and alimentary tissues it was shown that the nose,
trachea, and lungs, had about equally high activities on a per organ
basis (5-7 nmol/hour), whereas all other tissues had considerably
less activity (Dahl et al., 1985).
2.1.2.2. In vivo
When rats were given 14C-B(a)P in oral doses of 10.2, 102, or
1020 µg/rat, 74-79% of the dose was excreted in the faeces in the
first 48 hours. Unchanged B(a)P in faeces amounted to 13.0, 7.8,
and 5.6%, respectively, for the three doses given. Faecal
metabolites included 3-OH-B(a)P, 9-OH-B(a)P, B(a)P-3,6-quinone,
B(a)P-1,6-quinone, and trace amounts of B(a)P-4,5-dihydrodiol, and
B(a)P-7,8-dihydrodiol. When rats were fed charcoal-broiled
hamburger containing 52.7 µg B(a)P/kg, 11% (0.06 µg/rat) of the
B(a)P consumed was excreted unchanged in the faeces. When humans
ate meals containing charcoal-broiled meat (24.2 µg B(a)P/kg) B(a)P
was not detected in the faeces, despite the fact that each person
consumed 8.6 µg B(a)P (Hecht et al., 1979).
B(a)P was administered orally to Wistar rats at doses of 10, 20
and 50 umol/kg body weight/day 3 consecutive days. Urine was
collected for a total of 6 days. At all dose levels, the urinary
excretion of 3-OH-benzo[a]pyrene amounted to approximately 0.3% of
dose. Three other metabolites were seen in urine, but not
identified (Jongeneelen et al., 1984).
3-OH-B(a)P and mutagenic activity in rat urine were determined
after oral administration of B(a)P given in three repeated doses of
10, 20 and 50 µmol/kg/bw. The mutagenic activity of concentrated
urine samples was assayed with the Salmonella typhimurium strain
TA98 in the presence of S9 mix and ß-glucuronidase. The urinary
excretion of 3-0H-B(a)P and mutagens showed a correlation and both
increased dose-dependently during the sampling period of 6 days
(Jongeneelen et al., 1985).
Female Lewis rats administered 3-OH-B(a)P (50 mg/kg,
intraperitoneally) excreted metabolites via the bile. After
treatment with ß-glucuronidase and aryl sulphatase a minor, highly
labile metabolite, tentatively identified as 3,5-dihydroxy-B(a)P,
was found in addition to 3-OH-B(a)P-7,8-dihydrodiol and 3-OH-B(a)P
(Ribeiro et al., 1985).
When rats (germfree and conventional) were dosed with 14C-
B(a)P, a large part of the metabolites (9-24% depending on animal
type and route of excretion) had amphoteric properties, like
glutathione and cysteine conjugates. The abundance of conjugates
sensitive to ß-glucuronidase and sulphatase was low. The relative
amount of acidic conjugates in faeces was much higher in the
germfree than in the conventional rats, indicating the influence of
the intestinal flora on the metabolism. The results support the
view that the mercapturic acid pathway is a quantitatively important
route for B(a)P in rats (Egestad et al., 1987).
Groups of male Fisher F344 rats were fed cooked, low-fat human
diets and given 14C-B(a)P (0.6 µmol) by gavage. Potential reactive
metabolites in the gastrointestinal tract were trapped with magnetic
polyethyleneimine microcapsules. Approximately 70% of the dose was
recovered in the faeces when the diet had a low fibre content, while
80% was excreted in the faeces when the diet had a high fibre
content. One to two % of dose was bound to the microcapsules. The
metabolites found bound to microcapsules were B(a)P-3,6-quinone,
B(a)P-1,6-quinone, an unidentified metabolite, and B(a)P-tetraols.
Addition of beef protein to the diet increased the amount of
metabolites bound to the microcapsules (O'Neill et al., 1990a;
1990b).
Approximately one-third of an intravenous dose of 14C-B(a)P
was excreted within 4 hours in the bile of guinea-pigs fed a normal
diet. The extent of excretion was not altered by feeding high-fat
(17.3% coconut oil) or high-cholesterol (0.1% in 17.2% coconut oil)
diets. Hepatic cytochromes P-450 and b5, and B(a)P-hydroxylase
activity were unaltered by the administration of high-fat and high-
cholesterol diets. Pretreatment with low oral doses of B(a)P (6 X 3
mg/kg body weight) did not induce these parameters in animals given
any of the diets. High-fat and high-cholesterol diets altered the
pattern of B(a)P metabolites in the bile, with significantly
increased excretion of dihydrodiol glucuronides in both the high-fat
and high-cholesterol groups. Hepatic epoxide hydrolase activity and
glutathione content were unaltered by the high-fat or high-
cholesterol diets, and therefore cannot explain the alteration in
the profile of biliary metabolites of B(a)P. The altered pattern of
biliary excretion in animals fed high-fat or high-cholesterol diets
would lead to an increase in the delivery to the colon of
dihydrodiol metabolites of benzo[a]pyrene (Bowes & Renwick, 1986a).
Strains of intestinal bacteria from guinea-pigs were capable of
deconjugating B(a)P metabolites in vitro. The hydrolysis
products, and other primary oxidative metabolites of B(a)P, were
stable to further degradation by the strains tested. B(a)P
hydroxylase was measurable in the mucosa of the upper intestine, but
was present in the lower gut only at very low levels in some
animals. The activity was inducible, by oral administration of
B(a)P, in small intestinal mucosa of guinea-pigs fed normal diet but
not in those fed high-fat and high-cholesterol diets. Low levels of
covalent binding of 3H to DNA of liver and gut mucosa were obtained
in guinea-pigs dosed orally with 3H-B(a)P (app. 100 µg). The
feeding of high-fat and high-cholesterol diets did not increase this
binding. Guinea-pigs fed high-fat and high-cholesterol diets
excreted a greater proportion of an oral dose of 3H-B(a)P in urine,
and less in faeces, than animals fed a normal diet (Bowes & Renwick
1986b).
2.1.3 Effects on enzymes and other biochemical parameters
B(a)P and other polycyclic aromatic hydrocarbons (PAHs)
stimulate their own metabolism by inducing microsomal cytochrome P-
450 monoxygenases and epoxide hydrolase. This has been most
thoroughly studied with 3-methylcholanthrene (3-MC). The most
significant isozymes induced belong to the cytochrome P450IA
subfamily, namely P450IA1 (P4501 in the mouse; P450c in the rat)
which is the major isozyme induced and P450IA2 (P450d in the rat).
The induction is mediated by binding to a cytosolic receptor
protein, the Ah receptor, and the receptor-inducer complex is
translocated to the cell nucleus where its binding to a promoter
sequence in DNA triggers the transcription of genes producing the
enzymes. The Ah receptor is genetically controlled by the Ah locus.
Strains of mice (e.g. B6) having high affinity receptors are readily
induced (responsive mice; Ahb/Ahb), while other strains (e.g. C3 and
D2) having low affinity receptors are much less prone to induction
(non-responsive mice; Ahd/Ahd). When the induction is measured in
vitro in microsomes using B(a)P as the substrate, the activity is
termed aryl hydrocarbon hydroxylase (AHH) or B(a)P hydroxylase
(B(a)PH) activity, and is primarily a determination of the 3-
hydroxylation of B(a)P. The AHH activity is inducible not only in
the liver, but also in most extrahepatic tissues. Numerous studies
have shown that AHH induction not only leads to an enhanced turn-
over of B(a)P, but also lead to enhanced generation of the active
metabolites (presumably diolepoxides) that bind to cellular
macromolecules, and induce mutations and cancer (Nebert & Jensen,
1979; Gelboin, 1980; Conney, 1982; Cooper, 1983).
In the pregnant Sprague-Dawley rat the in vivo metabolism of
3H-B(a)P was markedly increased when the rats had been pretreated
for three days with 10 mg B(a)P/kg body weight/day or higher doses.
This treatment also induced AHH activity in the placenta and the
fetal liver (Welch et al., 1972).
The in vitro liver microsomal AHH activity was significantly
increased in rats 24 hours after intraperitoneal doses of 4 mg
B(a)P/kg body weight and 48 hours after 2 mg B(a)P/kg body weight,
but not after 1 mg B(a)P/kg body weight. The in vivo binding of
3H-B(a)P to liver DNA showed linear dose-response relationship in
the dose range 40 µg to 1 mg B(a)P/kg body weight, followed by a
step towards 2 mg/kg, and a shallow linear slope above that value.
The binding to liver DNA from an equimolar dose of B(a)P was 35
times less than the binding found in mouse skin (Lutz et al.,
1978).
Groups of 6 female Sprague-Dawley rats were fed a fibre-free
purified diet for 7 days, then they were switched to experimental
diets for 48 hours. After another 48 hours, the small intestinal
mucosa was assayed for B(a)P hydroxylase (B(a)PH) activity.
Experimental diets contained 0, 100, 400, 800, or 1200 mg B(a)P/kg
diet each with and without 10% soft white wheat bran. Enzyme
induction with 100 and 400 mg B(a)P/kg diet was partially inhibited
by bran, but with higher concentrations of B(a)P there was no
protective effect. The inhibition in B(a)P-induced B(a)PH activity
was observed with 10% wheat bran but not with 3.3 or 6.6%.
Subsequent studies showed no significant inhibition in B(a)PH
induction with cellulose or lignin, whereas all forms of wheat bran
(hard red, soft white, or finely ground soft white) caused
significant inhibition. A diet containing charcoal-broiled beef was
compared with diets containing raw beef or soybean protein each with
and without 10% soft white wheat bran. B(a)PH activity remained low
with raw beef and soybean protein whether or not fiber was added.
However, intestinal B(a)PH activity was raised ninefold by charcoal-
broiled beef. The addition of bran reduced B(a)PH activity to 65%
of that observed with the fibre-free, charcoal-broiled beef diet
(Clinton & Visek, 1989).
B(a)P hydroxylase (B(a)PH) activity was measured in homogenates
of fetal liver (day 18) or of whole embryos of mice on day 9, 10 or
12 of gestation after oral maternal pretreatment with B(a)P on 3
consecutive days. Three oral doses of 17.5 mg B(a)P/kg body weight
were found to just significantly induce B(a)PH in maternal liver.
An induction was observed after pretreatment with 24 mg B(a)P/kg
body weight in 9-, 10- or 12-day-old whole embryos. The induction
was demonstrable in embryos at tissue levels about one order of
magnitude lower than those required for induction in maternal liver.
Treatment with 25 mg B(a)P/kg body weight on 3 consecutive days was
required to induce B(a)PH in fetal liver on day 18 of gestation.
The required B(a)P tissue concentrations were about one half of
those necessary for induction in maternal liver (Neubert & Tapken,
1988).
2.2 Toxicological studies
2.2.1 Acute toxicity studies
The LD50 in the mouse after intraperitoneal injection is about
250 mg/kg body weight (IARC, 1983).
2.2.2 Short-term studies
See special studies.
2.2.3 Long-term/carcinogenicity studies
B(a)P is a well documented carcinogen producing mainly local
tumours in a variety of species after skin application, inhalation
and/or intratracheal administration, intrabronchial implantation,
subcutaneous and/or intramuscular administration, and a variety of
other applications. Tumours are also induced after intraperitoneal,
intravenous, and transplacental injection. Studies showing
carcinogenicity after oral administration have also been reported.
B(a)P appears to be a less potent carcinogen after systemic
administrations than after local applications (IARC, 1973; 1982;
1983).
The following is a brief summary of carcinogenicity studies
that have used mainly oral administration of B(a)P.
2.2.3.1 Mouse
Forestomach
Groups of 20 female albino mice were treated with single
intragastric dose of 0.2 mg B(a)P/mouse (corresponding to 6 mg/kg
body weight). After 43 weeks a total of 14 tumours in the
forestomach was observed in 5 of 11 surviving animals. Single doses
of 0.05 mg (1.5 mg/kg body weight) and 0.012 mg (0.36 mg/kg body
weight) produced 0/9 and 2/10 tumours, respectively. No tumours
were seen in 9 surviving control animals. Weekly treatment with
lime oil enhanced the tumour incidence. No tumours were seen in the
glandular part of the stomach or other parts of the alimentary canal
(Pierce, 1961).
White Swiss mice fed 250 or 1000 ppm of B(a)P in the diet for
various time periods developed squamous papillomas and carcinomas of
the forestomach in a dose dependent manner. All mice fed 1000 ppm
B(a)P and examined after 86 days had tumours. In the group fed 250
ppm B(a)P 25% developed gastric tumours when fed for more than 85
days. A high incidence of lung adenomas was also observed in these
mice (Rigdon & Neal, 1966).
No increases in stomach tumours were found in mice after 110
days of treatment with diets containing up to 30 ppm B(a)P, while
40-45 ppm for 110 days induced stomach tumours in about 10% of the
mice, and more than 70% of mice fed 50-250 ppm for 122-197 days had
stomach tumours. A diet containing 250 ppm B(a)P fed for different
time periods produced the following incidences of stomach tumours;
one day, 0%; 2-4 days, 10%; 5-7 days, 30-40%; 30 days, 100% (Neal &
Rigdon, 1967).
Groups of 19-20 female Ha/ICR mice were fed 300 ppm or 100 ppm
B(a)P in their diets for 17 and 31 weeks, respectively. The
percentages of mice with forestomach tumours were 68% and 55%,
respectively. In 12 female A/HeJ mice fed 1000 ppm B(a)P in their
diet for 12 weeks all mice developed tumours of the forestomach
(Wattenberg, 1972).
Groups of 20 female Ha/ICR mice (9 weeks old) were fed a diet
containing 300 ppm B(a)P for 6 weeks and thereafter maintained on
control diet for 14 weeks. Forty percent of the mice developed
forestomach tumours. The tumours were inhibited by concurrent
feeding with disulfiram (Wattenberg, 1974).
In a similar experiment where 17 female Ha/ICR mice 9 weeks of
age were fed 300 ppm B(a)P for 6 weeks followed by 14 weeks on
control diet 41% of the mice developed forestomach papillomas. The
tumours were completely inhibited by benzyl isothiocyanate added to
the diet (Wattenberg, 1977).
Groups of 9 female Ha/ICR mice (9 weeks old) were fed diets
containing 200 or 300 ppm B(a)P for 12 weeks. In the 200 ppm group
66%, and in the 300 ppm group 100% of the mice had forestomach
papillomas. AHH activity was elevated in the forestomach, glandular
stomach and lung, but not liver of these mice (Triolo et al.,
1977).
Eight doses of 1.5 mg B(a)P were administered twice a week for
four weeks to 20-24 ICR mice (5 weeks old). After 25 weeks the
average number of forestomach papillomas per mouse was approximately
5. Nitrite (0.05% in drinking-water) and soy sauce (20% in a
refined diet) together significantly reduced the number of neoplasms
per animal (Benjamin et al., 1988).
Eight organosulfur compounds from garlic and onions were
studied for their inhibitory effects on B(a)P-induced neoplasia of
forestomach and lung of female A/J mice when administered 96 and 48
hours prior to carcinogen challenge. B(a)P (2 mg/mouse) was given
perorally 3 times with two week intervals to groups of 15 mice. The
study was terminated 26 weeks after the first B(a)P dose.
Approximately 3 papillomas per mouse were seen in the forestomach
and approximately 15 adenomas per mouse in the lung after B(a)P.
Allylic compounds inhibited B(a)P-induced neoplasia of the
forestomach while the saturated analogs were almost without
inhibitory activity. All the allylic compounds induced increased
glutathione S-transferase (GST) activity in the forestomach, but
varied in their capacity to induce GST in lung, liver and small
bowel. Their saturated analogs produced little or no induction
(Sparnins et al., 1988).
Nomilin, a limonoid found in edible citrus fruits and an active
inducer of glutathione S-transferase activity in the liver and small
intestinal mucosa of female ICR/Ha mice was found to inhibit
perorally B(a)P-induced (1 mg per animal twice a week for 4 weeks)
neoplasia in the forestomach. The number of mice with tumours after
18 weeks was reduced from 100 to 72%, and the number of tumours per
mouse was significantly decreased as a result of nomilin treatment
(Lam & Hasegawa, 1989).
Lung tumours
The induction of lung adenomas and leukaemia in mice after 140
days on a diet containing 250 ppm B(a)P has been reported (Rigdon &
Neal, 1969).
The induction of lung adenomas after peroral administration of
B(a)P was confirmed in groups of 15 female A/HeJ mice. B(a)P was
given by intubation at a dose of 6 mg B(a)P (2x3 mg B(a)P), and
repeated after 3 weeks. After 19 weeks an average of 16 pulmonary
adenomas per mouse was found compared to 0 in the controls. The
induction of lung tumours was almost abolished by treatment of the
mice with beta-naphthoflavone (an inhibitor of cytochrome P4501)
for 3 weeks prior to B(a)P (Wattenberg & Leong, 1970).
All 24 female A/HeJ mice given two doses of 3 mg B(a)P/mouse by
gavage at 14 day interval developed pulmonary adenomas after 20
weeks (Wattenberg, 1973).
All 12 female A/HeJ mice (10 weeks old) given two peroral
intubations with 3 mg B(a)P/mouse with two week interval and
maintained on control diet for another 21 weeks developed lung
adenomas (100%; 7.8 tumours/mouse). Disulfiram did not inhibit
tumour induction (Wattenburg, 1974).
A single intraperitoneal dose of 100 mg/kg body weight of BaP
to groups of 20 male A/J mice produced pulmonary adenomas in all
mice within 6 months. The average number of tumours per mouse was
10.2. Three phenolic compounds (ferulic, chlorogenic and ellagic
acids) inhibited markedly the number of tumours per mouse but not
the number of mice with tumours (Lesca, 1983).
Leukaemias
Oral B(a)P doses estimated to be between 6 and 12 mg/kg body
weight/day induced leukaemia in non-responsive (Ahd/Ahd) mice after
100 or more days, but not in responsive mice (Ahb/Ahd). It was
suggested that a higher dose is obtained in the bone marrow of non-
responsive mice than in responsive mice (Nebert & Jensen, 1978).
When given to 42-day old mice a single intraperitoneal
injection of 75-100 µg B(a)P/kg body weight produced a high
frequency of lymphoreticular tumours after 90 weeks. When B(a)P was
given to either 1-day or 8-day old mice the tumour incidences were
lower (Vesselinovitch et al., 1975).
Intracolonical administration
Groups of 50 male and 50 female Swiss mice (6 weeks old)
received one or 10 intracolonical instillations of 200 µg B(a)P/g
body weight. The treatment had no effect on survival of the mice
compared to controls. The single administration of B(a)P induced
malignant lymphomas (42% in females, 12% in males compared to 14%
and 0% in controls) and forestomach tumours (10% in females, 4% in
males compared to 2% and 0% in controls). In the mice given 10
doses of B(a)P neoplasia of oesophagus (10% in females, 0% in males;
2% and 0% in controls), anus (12% in females, 10% in males; 0% and
0% in controls), and skin (22% in females, 26% in males; 0% and 2%
in controls) were seen in addition to malignant lymphomas (36% in
females; 14 in males) and forestomach tumours (22% in females; 20%
in males) (Toth, 1980).
Groups of 45 female Ha/ICR mice and 38 female C57B1/6 mice were
given 1 mg B(a)P/mouse intrarectally once weekly for 14 weeks, and
thereafter maintained on control diet until 18 months. In the
Ha/ICR mice 27/37 (73%) developed primary lung tumours (25% in
control mice) and 16/17 (94%) mice had forestomach tumours (20% in
controls). Mammary tumours occurred in 23% of the treated mice
compared to 9% in the control mice. In the C57B1/6 mice 94%
developed forestomach tumours (21% in controls), 28% lymphomas (2%
in controls), and 16% peritoneal sarcomas (0 in controls), 28%
lymphomas (2% in controls), and 16% peritoneal sarcomas (0 in
controls). No colonic neoplasms were found in any of the mice after
18 months (Anderson et al., 1983).
Newborn mouse
Subcutaneous or intraperitoneal injection of 20-40 µg
B(a)P/mouse during the first day of life have produced lung adenomas
and/or hepatomas in different strains of mice after 50-60 weeks.
Malignant lymphoma and mammary adenocarcinoma have also been
reported (Peitra et al., 1961; Roe & Waters, 1967; IARC, 1973).
Intraperitoneal administration of a total dose of 1400 nmol
B(a)P-7,8-dihydrodiol/mouse to newborn Swiss-Webster mice on days 1,
8, and 15 of life produced more malignant lymphomas and pulmonary
adenomas than the equimolar dose of B(a)P. B(a)PDEI ((+)-anti-
B(a)PDE) produced the same incidence of pulmonary adenomas at a much
lower dose (28 nmol/mouse) (Kapitulnik et al., 1977; 1978). Of
the four optical enantiomers of the diastereoisomeric B(a)P-7,8-
diol-9,10-epoxides only B(a)PDEI had an exceptional pulmonary
tumorigenicity in newborn Swiss-Webster mice when given in total
doses of 7 (71% of mice with tumours) or 14 nmol (100% of mice with
tumours) (Buening et al., 1978).
Transplacental route
Subcutaneous injection of 2-4 mg B(a)P to pregnant ICR/Ha mice
on days 11, 13, and 15 of the pregnancy resulted in an increased
incidence of lung adenomas (62%) in the offspring when they were 28
weeks of age. The treatment also increased skin carcinogenesis of
B(a)P in the offspring (Bulay & Wattenberg, 1970).
Direct injection of B(a)P, B(a)P-4,5-oxide (4-20 nmol/foetus),
and B(a)PDE (racemic mixture) (0.4-4 nmol/foetus) to fetal Swiss
mice on day 15 of intrauterine growth produced pulmonary adenomas at
12-15 weeks of age (Rossi et al., 1983).
2.2.3.2 Rat
Nine female Sprague-Dawley rats (50-65 days of age) were given
a single oral dose of 100 mg B(a)P. Within 60 days, 8/9 rats
developed tumours of the mammary gland (papillary adenocarcinomas)
(Huggins & Yang, 1962).
In a study with Sprague-Dawley rats of both sexes (3 1/2 months
of age) daily doses of 2.5 mg B(a)P/rat for 8-12 months induced
papillomas of the oesophagus and forestomach in 3 out of 40 animals
(Gibel, 1964).
B(a)P was administered for 87-131 weeks to groups of 32 male
and 32 female Sprague-Dawley rats either as an admixture to the diet
or by gavage in an aqueous 1.5% caffeine solution. B(a)P in
solution was given as doses of 0.15 mg/kg bw either: 1) 5 days per
week (annual dose: 39 mg/kg bw ), 2) every 3rd day (18 mg/kg
bw/year), 3) or every 9th day (6 mg/kg bw). When mixed in the diet
the doses were: 4) 0.15 mg/kg body 5 days a week (39 mg/kg bw/year)
or 5) 0.15 mg/kg bw every 9th day (6 mg/kg bw/year). Similar groups
given either caffeine solution or control diet served as controls.
A significant increased number of rats with forestomach papillomas
was observed in group 1 to 4 (14, 25, 11, and 9 rats with tumours,
respectively) compared to 1 in group 5, and 2 and 3 tumours in the
control groups. No other tumours were found significantly different
from control levels (Brune et al., 1981).
Intraperitoneal administration of B(a)P (50 mg/kg bw) 18 hours
after partial hepatectomy induced enzyme-altered foci in rats
subsequently promoted with 2-acetylaminofluorene/CC14.
Pretreatment of rats with 3-MC 66 hours before partial hepatectomy
and 84 hours before B(a)P administration, increased the number of
enzyme-altered foci (Dock et al., 1988).
2.2.3.3 Hamster
Bi-weekly administration of 2-5 mg B(a)P by gavage produced 5
papillomas of the stomach in 67 hamsters treated for one to five
months, seven papillomas and two carcinomas in 18 hamsters treated
for six to nine months and five papillomas in eight hamsters treated
for 10-11 months (Dontenwill & Mohr, 1962). In another experiment a
diet containing 500 ppm B(a)P was given to 13 hamsters four days per
week for up to 14 months. A total of 12 tumours (two in oesophagus,
eight in the forestomach and two in the intestine) were seen in
eight hamsters (Chu & Malmgren, 1965).
A low incidence of large bowel neoplasms was induced in a group
of 30 male Syrian hamsters of the inbred strain BIO15.16 by
intrarectal instillation of B(a)P. The hamsters were given 0.8 mg
B(a)P once weekly for 30 weeks. The experiment was terminated after
1 year. The incidence of colon neoplasms was 6% with B(a)P exposure
(2/30 adenocarcinomas versus 0/15 in control hamsters) (Wang et
al., 1985).
2.2.2.4 Reproduction studies
Oral B(a)P (120 mg/kg/day) given to pregnant Ahd/Ahd mice (non-
responsive mice) between gestational days 2 and 10 produced more
intrauterine toxicity and malformations in Ahd/Ahd (non-responsive)
than in Ahb/Ahd (responsive) embryos. Pharmacokinetic studies with
3H-B(a)P in the diet showed that in the pregnant Ahd/Ahd mouse
little induction of B(a)P metabolism occurred in the intestine and
liver, leading to much larger amounts of B(a)P reaching the embryos.
In the pregnant Ahb/Ahd mouse B(a)P metabolism was greatly enhanced
in the intestine and liver; this led to less B(a)P reaching the
embryos, and much less intrauterine toxicity and malformations.
More toxic metabolites (especially B(a)P 1,6- and 3,6-quinones) were
shown to occur in Ahd/Ahd embryos than in Ahb/Ahd embryos
(Legraverend et al., 1984).
B(a)P (50 mg/kg bw) was given subcutaneously to pregnant rats
at different stages of gestation. B(a)P affected the reproductive
performance of pregnant rats by significantly increasing the number
of resorptions and fetal wastage, and by decreasing the fetal weight
(Bui et al., 1986).
The teratogenicity of B(a)P, B(a)P-4,5-oxide, and a racemic
mixture of B(a)PDEI was investigated after direct (intrauterine)
injection into embryonal Swiss mice. The compounds were injected at
doses ranging from 0.4 to 16.0 nmol/embryo on days 10, 12, and 14 of
development. The transplacental effects of B(a)P given at the same
gestational days and a comparable dose level (47.5 µmol/kg bw) were
also evaluated. The fetuses were examined when they were 18 days
old. On the basis of gross external and internal malformations,
B(a)PDEI appeared to be the most potent embryotoxic and teratogenic
compound tested, causing 85% embryolethality and 100% malformed
fetuses in the group treated on day 10 (0.4 nmol/embryo) of
intrauterine development. There were 61 and 27% of fetuses
malformed following B(a)PDEI treatment on days 12 and 14 of
gestation, respectively (2 and 4 nmol/embryo). The effects of this
B(a)P metabolite were very specific and malformations such as
exencephaly, thoraco- and gastroschisis, phocomelia, and oedema were
found. The administration of B(a)P (both transplacental and direct
intraembryonal injection) and B(a)P-4,5-oxide caused no significant
increase of malformed foetuses in any of the developmental stages
considered (Barbieri et al., 1986).
Mouse endometrial cell microsomes showed inducible cytochrome
P-450 mediated oxidation of B(a)P(-)-trans 7,8-dihydrodiol to B(a)P-
7,8-dihydrodiol 9,10-epoxides. Embryos obtained from pregnant mice
on day 3 post-coitum were incubated with endometrial cell microsomes
and B(a)P(-)-trans-7,8-dihydrodiol at various concentrations from 0
to 1.0 µM. Following the incubation, the embryos were transferred
to pseudopregnant surrogate mothers which were sacrificed 7 days
later. The number of surrogate mothers remaining pregnant following
transfer was reduced significantly at the highest concentration of
B(a)P(-)-trans-7,8-dihydrodiol. Blastocyst implantation and
decidual swelling volume was reduced in a concentration dependent
manner (Iannaccone et al., 1984).
2.2.5 Special studies on genotoxicity
B(a)P has been extensively studied for mutagenic activity and
is used as a positive control in a variety of short-term tests. It
was active in assays for bacterial DNA repair, bacteriophage
induction and bacterial mutation; mutation in Drosophila
melanogaster; DNA binding, DNA repair; sister chromatid exchange,
chromosomal aberration, point mutation and transformation in
mammalian cells in culture; and tests in animals in vivo,
including DNA binding, sister chromatid exchange, chromosomal
aberration, sperm abnormality and the somatic specific locus (spot)
test (IARC, 1983).
In vitro
Isolated monkey hepatocytes were more efficient than human
hepatocytes in their capacity to activate B(a)P into products
mutagenic towards Salmonella typhimurium TA 1538. It was shown
that monkey liver preparations seem to possess a higher
monooxygenase activity towards B(a)P than human liver preparations
(Neis et al., 1986).
B(a)P was assayed for mutagenicity in the Ames test, in the
presence of hepatic post-mitochondrial preparations isolated from
the mouse, rat, hamster, pig and man. B(a)P gave a positive
mutagenic response only in the presence of activation systems
derived from the hamster (Phillipson & Ioannides, 1989).
No mutagenic metabolites of B(a)P were detected in the
perfusate from an isolated perfused rat-liver system using either
the Ames test or a bioluminescence test for genotoxic agents. The
bile showed strong genotoxic activity especially in the presence of
the deconjugation enzymes ß-glucuronidase and arylsulfatase (Ben-
Itzhak et al., 1985).
Liver microsomal enzymes from male Sprague-Dawley rats
pretreated with Aroclor 1254 were more effective in inducing B(a)P-
mediated mutagenesis in the Salmonella/mammalian microsome
mutagenicity test than microsomes from DDT treated rats. DDT-
induced microsomes yielded a greater proportion of B(a)P-4,5-oxide
and its metabolic product B(a)P-4,5-dihydrodiol than did Aroclor-
induced microsomes (Bonin et al., 1985).
Rat small-intestinal microsomes were compared with liver
preparations for their ability to activate B(a)P using the
Salmonella mutagenicity assay. At lower doses (less than 1 µg
B(a)P/plate) comparatively high levels of activation were obtained
with intestinal microsomes. This could be due to preferential
formation of the mutagenic 4,5-oxide with the intestinal microsomes,
as opposed to the putative major active metabolite, the 7,8-diol-
9,10-epoxide (Walters & Combes, 1986).
Liver microsomes from rats with vitamin A deficiency enhanced
the mutagenicity of B(a)P in Salmonella typhimurium TA 98 (Alzieu
et al., 1987).
Selenium inhibited the S9 dependent mutagenicity of B(a)P and a
number of its metabolites to Salmonella typhimurium strain TA100.
The results suggested that selenium modified the metabolism and
hence the mutagenicity of B(a)P to TA100 by affecting mixed-function
oxidase and/or epoxide hydratase activity in both the rat and
hamster liver S9 activation systems. Differences were reflected in
decreased amounts of the strongly mutagenic B(a)P-7,8-dihydrodiol
and increased amounts of 4,5- and 9,10-dihydrodiols that were weakly
mutagenic (Teel, 1984).
Selenium (sodium selenite) decreased the mutagenicity of B(a)P
in Salmonella typhimurium strains TA98 and TA1000 (Prasanna et
al., 1987).
Small amounts of seminal fluid strongly enhanced the
mutagenicity of B(a)P in the Salmonella/microsome test. The
effect was found only in the presence of S9 mix for metabolic
activation (Rivrud, 1988).
Using different scavengers of active oxygen species (superoxide
dismutase, catalase, mannitol and dimethylfuran) in the Ames
Salmonella assay to determine the role of the reactive oxygen
species in the B(a)P mutagenesis process, it was suggested that
singlet oxygen (inhibited by dimethylfuran) may play an important
role in promoting B(a)P mutagenicity (Wei et al., 1989).
The mutation frequency (resistance to ouabain) induced by B(a)P
was substantially inhibited dose dependently by haemin in Chinese
hamster V79 cells co-cultivated with X-irradiated hamster embryo
cells. The mutagenicity of B(a)P (1 microgram/ml) on V79 cells was
reduced 6.5% by haemin, 52% by biliverdin, 73% by protoporphyrin and
85% by chlorophyllin (Katoh et al., 1983).
Studies on the cytotoxicity and mutagenicity to 6-thioguanine
resistance by S9-activated B(a)P in asynchronized and synchronized
Chinese hamster V79 cells suggested the presence of a specific hot
spot in the cell cycle for mutagenesis by the B(a)P in cultured
hamster cells (Ochi et al., 1985).
Irradiated second passage Wistar rat embryo (WRE) cells were
used as activator cells for B(a)P; V79 Chinese hamster lung cells
were used as the target cells and exposed to 3H-B(a)P for 5, 24 and
48 hours under the conditions of a cell-mediated mutation assay. A
correlation was found between mutation induction and the amount of
B(a)PDEI-deoxyguanosine (dGuo) adduct in the V79 cells (Sebti et
al., 1985).
The triol 3-OH-B(a)P-7,8-diol was not mutagenic in Salmonella
typhimurium strains TA 97, TA 98, TA 100 or TA 1537 or in V79
Chinese hamster cells (6-thioguanine) when no exogenous metabolizing
system was added. In the presence of S9 mix, the triol was 5-18
times more mutagenic than 3-OH-B(a)P in strains TA 97, TA 100 and TA
1537, but both compounds showed similar mutagenic potencies with
strain TA 98. B(a)P-7,8-diol, like the triol, showed mutagenic
effects only in the presence of S9 mix. In V79 cells, the diol was
a potent mutagen, while the triol showed only very weak mutagenic
effects (Glatt et al., 1987).
B(a)P did not effect development and induction of sister
chromatid exchanges (SCEs) in cultured mouse blastocysts when added
at a concentration of 1 uM (Spindle & Wu, 1985).
Induction of SCE in the mouse hepatoma cell line TAOc1B(a)Prc1
by B(a)P was due to the production of B(a)P-7,8-dihydrodiol. This
metabolite did not appear to be produced by another mouse hepatoma
cell line B(a)Prc1. TAOc1B(a)Prc1 required only 40 nM B(a)P to
induce a 2-fold increase in SCE frequency (Schaefer & Selkirk,
1985).
B(a)P-dependent mutagenesis was strongly inhibited in a
concentration-dependent manner by 7,8-benzoflavone in a murine
keratinocyte cell-mediated mutagenesis assay (Reiners, 1985).
B(a)P was mutagenic and induced chromosomal aberrations in the
L5178Y/TK+/- mouse lymphoma assay and induced sister chromatid
exchanges in vivo using the mouse peripheral blood lymphocyte
culture system (Klingerman et al., 1986).
A recombinant plasmid containing the thymidine kinase (TK) gene
(pAGO; 6.36 kilobases) was reacted in vitro with B(a)PDEI. Upon
transection of mouse LTK-cells with modified plasmid or modified TK
gene, none or only a few TK-positive cells were obtained, in
contrast to the formation of many colonies after transection with
the unmodified plasmid (gene), B(a)P itself, and phenanthrene oxide,
a weakly reactive but noncarcinogenic chemical (Schaefer-Ridder et
al., 1984). The genomic level of DNA cytosine methylation was
significantly diminished in dividing BALB/3T3 CL1-13 cells treated
with B(a)P. The decrease in DNA 5-methylcytosine levels was
concentration dependent over the range of 0.1 to 1.0 µg/ml when
determined at the end of the 16 hour treatment period. (+/-)-SYN-
B(a)PDE was implicated as the active metabolite causing the effect
(Wilson & Jones, 1984).
Suspensions of rat colon epithelial cells metabolized B(a)P
into products mutagenic for human P3 teratoma cells. Mutagenesis in
the P3 cells was indicated by an acquired resistance to 6-
thioguanine (Oravec et al., 1986).
B(a)P was cytotoxic and enhanced viral transformation in the
Syrian hamster embryo/simian adenovirus SA7 (SHE/SA7) viral
enhancement assay (Lubet et al., 1986).
The effects of glucuronide conjugation on B(a)P-induced
cytotoxicity and mutagenicity were studied using the CHO/HGPRT assay
with a rat liver homogenate preparation containing Mixed Function
Oxidase (MFO) system cofactors (S9 mix) and uridine diphosphate a-D-
glucuronic acid (UDPGA). A reduction of B(a)P and B(a)P 6-OH-
induced cytotoxicity of glucuronide conjugation was probably due to
the elimination of cytotoxic phenols and quinones. Since B(a)P 7,8-
diol is a poor substrate for UDP-glucuronyltransferase enzymes, no
effects on B(a)P-induced mutagenicity or B(a)P 7,8-diol-induced
cytotoxicity and mutagenicity were observed (Recio & Hsie, 1984).
Arachidonic acid (AA), a prostaglandin precursor, significantly
potentiated sister-chromatid exchange (SCE) induction in vitro by
B(a)P in the aryl hydrocarbon hydroxylase (AHH)-inducible human
hepatoma C-HC-4 cells, and to a lesser extent in the non-inducible
rat tumour AH66-B and R1 and Chinese hamster Don-6 cells, all of
which were less sensitive than C-HC-4 cells. Indomethacin
completely eliminated the potentiating effect of AA on SCE induction
by B(a)P (Abe, 1986).
A continuous B(a)P dose as low as 0.02 µM for 20 days produced
a significant increase in mutant fraction at the 6TG-resistance
(HGPRT) locus in metabolically competent human lymphoblastoid cells.
The long-term, low-dose protocol (0-1 µM for up to 20 days) was
significantly more efficient at inducing mutations than a short-
term, high-dose protocol (0-10 µM for 1 day) (Danheiser et al.,
1989).
The active metabolite of B(a)P, B(a)PDEI caused cytotoxicity
and induced mutations in normally repairing or nucleotide excision
repair-deficient diploid human fibroblasts. Mutations induced in a
defined gene sequence, supF, when plasmid containing adducts formed
by the compound replicated in human 293 cells were mainly base
substitution mutations, predominantly G:C to T:A transversions
(Maher et al., 1987).
In a cell-mediated mutagenesis assay, treatment of human
mammary epithelial cells with B(a)P resulted in significant rates of
mutagenesis in cocultured V-79 cells. No such effect was found with
rat cells under identical conditions. The most significant
qualitative difference in B(a)P metabolism between the two species
was the ability of the rat, but not the human, mammary epithelial
cells to conjugate significant amounts of B(a)P to glucuronic acid.
Other aspects of carcinogen metabolism, including production of the
precursors to known active metabolites of B(a)P were similar (Moore
et al., 1986).
In vivo
B(a)P and two of its major metabolites, B(a)P-4,5-oxide, and
B(a)P-7,8-diol were investigated for mutagenicity in Salmonella
typhimurium TA1538, TA98 and TA100 using an intrasanguineous host-
mediated assay. B(a)P and B(a)P-4,5-oxide were not mutagenic under
any experimental conditions. B(a)P-7,8-diol was inactive with the
strain TA1538 but was mutagenic with the strains TA98 and TA100.
The effect was potentiated by pretreatment of the host mice with the
cytochrome P-450 inducer 5,6-benzoflavone (Glatt et al., 1985).
B(a)P induced dose-related nuclear damage (micronuclei,
pyknotic nuclei and karyorrhectic bodies) in colonic epithelial
cells of female C57BL/6J mice within 24 hours when administered
intrarectally in single doses of 0-200 mg/kg body weight. This
damage was reduced when mice ingested the plant phenols, caffeic,
ferulic and ellagic acids, and quercetin at levels of 4% or BHA at
2% in the diet for 1 week prior to the B(a)P challenge (100 mg/kg
bw) (Wargovich et al., 1985).
Several organosulfur compounds in high concentrations, most
notably allyl mercaptan, benzyl mercaptan, and phenylethyl mercaptan
were active in inhibiting peroral B(a)P (100 mg/kg body weight)
nucleotoxicity to the colon of C57BL/6J mice (Wargovich & Eng,
1989).
B(a)P was tested in a micronucleus test using peroral
administration (62.5, 125, 250, and 500 mg/kg) to males of the MS/Ae
and CD-1 mouse strains. Initially, an acute toxicity study lasting
3 days was done to estimate LD50s. The LD50 was larger than 1600
mg/kg in the 2 strains. The full-scale micronucleus tests indicated
that B(a)P induced micronuclei dose-dependently in each strain
(Awogi & Sato, 1989).
Male B6C3F1 mice were injected intraperitoneally with B(a)P
(25, 75, 150, 300 mg/kg body weight). Twenty-four hours later
lymphocytes were cultured for analysis of SCE in B lymphocytes.
B(a)P induced significant dose-related increases in SCE frequency.
At the highest concentration B(a)P induced a 3.1-fold increase in
SCE frequency compared to concurrent controls (Kligerman et al.,
1985).
Male C57BL/6 mice and male Sprague-Dawley rats were injected
intraperitoneally with doses of B(a)P ranging from 10 to 100 mg/kg.
After 24 hours the peripheral blood lymphocytes (PBLs) were analyzed
for both DNA adduct formation by 32p-postlabeling and SCE induction
following lymphocyte culture. B(a)P induced similar, but not
identical, SCE induction following lymphocyte culture. B(a)P
induced similar, but not identical, SCE dose-response curves for
each species. After B(a)P administration, the major DNA adduct,
B(a)PDEI-dGuo, was approximately 10-fold more prevalent in the PBLs
of the mouse (300-1200 attomol/µg DNA) than those of the rat (20-130
attomol/µg DNA). Thus, for equivalent amounts of B(a)PDEI-dGuo, a
greater number of SCEs are induced in the rat than the mouse
(Kligerman et al., 1989).
B(a)P at 62.5 mg/kg body weight given by gavage to female Brown
Norway rats failed to induce unscheduled DNA synthesis in
parenchymal liver cells isolated 5 or 18 hours after the
administration. In contrast, alkaline elution showed that at 5
hours after administration of B(a)P a considerable number of alkali-
labile sites was present in the DNA of both intestinal cells and
parenchymal liver cells, but not in that of non-parenchymal liver
cells (Mullaart et al., 1989).
2.2.6 Special studies on macromolecular binding
The implication of covalent binding of B(a)P metabolites to DNA
for B(a)P carcinogenicity in skin and lung of experimental animals
has been intensively studied in cell culture systems and in vivo.
Several reviews have been published (Gelboin, 1980: Levin et al.,
1982; Pelkonen & Nebert, 1982; Conney, 1982; IARC, 1983; Cooper et
al., 1983; Grover, 1986). The metabolites responsible for binding
to DNA were the B(a)P-7,8-diol-9,10-oxide (B(a)PDEI) and the 9-OH-
B(a)P-4,5-oxide. Mutagenicity and carcinogenicity studies on a
variety of metabolites seem to indicate that B(a)PDEI is the
putative carcinogenic metabolite of B(a)P, although the correlation
between binding of this metabolite to DNA and tumour initiating
ability has not always proven perfect.
In vitro
B(a)P induced genotoxic effects and DNA adduct levels were
determined in several short-term bioassay systems: cytotoxicity,
gene mutation, and sister chromatid exchange in Chinese hamster V79
cells; cytotoxicity, gene mutation, and chromosome aberrations in
mouse lymphoma L5178Y TK+/-; cytotoxicity and enhanced virus
transformation in Syrian hamster embryo cells; and cytotoxicity and
morphological transformation in C3H10T1/2CL8 mouse embryo
fibroblasts. Both total B(a)P-DNA binding and specific B(a)P-DNA
adducts were measured. B(a)PDE I-dGuo was one of the major adducts
identified in all bioassay systems. DNA binding and genotoxic
responses varied significantly between bioassays. Each genetic end
point was induced with a differing efficiency on a per adduct basis.
However, the relationships between frequency of genetic effect or
morphological transformation and B(a)P-DNA binding or B(a)PDE I-dGuo
were linear within a given assay (Arce et al., 1987).
The cytochrome P-450-dependent metabolism of 3H-B(a)P by
cultured primary keratinocytes prepared from BALB/C mouse epidermis
was found to be largely inhibited by the dietary plant phenol,
ellagic acid. The intracellular enzyme-mediated binding of B(a)P to
mouse keratinocyte DNA was also largely inhibited in a dose-
dependent fashion (Mukhtar et al., 1984).
In vitro studies demonstrated that mouse serum sequesters
B(a)PDEI and protects it from hydrolysis. Four hours after B(a)P
administration to mice (i.p.), mouse serum produced two adduct spots
when incubated with salmon sperm DNA. The major adduct co-
chromatographed with a B(a)PDEI adduct standard. B(a)PDEI-DNA
adducts in tissues were highest in liver, lung and spleen, with
kidney and stomach levels significantly lower (Ginsberg & Atherholt,
1989).
The formation of 3H-B(a)P adducts with calf thymus DNA was
studied in vitro in the presence of microsomes prepared from the
isolated labyrinth zone of the rat placenta, the haematopoietic
erythroblast cells of the fetal liver, the fetal liver, as well as
the maternal liver. Pregnant rats were induced with ß-
naphthoflavone on day 17 of gestation and the microsomes prepared
one day later. The levels of covalent binding (pmol/mg DNA/mg
microsomal protein) for maternal liver, fetal liver, placenta and
erythroblast cells were: 28.4, 2.4, 0.31, and 3.9, respectively.
Major adducts were identified as the 9-OH-4,5-oxide adduct and the
B(a)PDEI adduct (Salhab et al., 1987).
Primary cultures of epithelial cell aggregates and fibroblasts
derived from mammary tissue from female Wistar rats able to
metabolize B(a)P and at least 7 DNA-adducts were isolated and
analyzed. None of the adducts showed chromatographic properties
characteristic of adducts formed by B(a)PDEI or other known
electrophilic metabolites of B(a)P. Similar profiles of adducts
were obtained from mammary DNA of rats that had been treated with
B(a)P by injection into their mammary fat pads. In contrast, when
B(a)P was administered by intraperitoneal injection to female Wistar
rats, B(a)PDEI-DNA adducts were detected in each of seven tissues,
including mammary gland, that were examined (Phillips et al.,
1985).
The covalent binding of B(a)P to calf thymus DNA brain
microsomes isolated from control and 3-methylcholanthrene (3-MC)
treated rats was investigated. Treatment of rats with 3-MC resulted
in a 1.5-fold increase in the brain microsomal mediated covalent
binding of 3H-B(a)P to DNA (Das et al., 1985b).
Using rat liver nuclei or hepatocytes incubated with B(a)P and
B(a)PDEI it was found that B(a)P binds more readily to DNA of active
chromatin and nuclear matrix than to bulk chromatin. Selectivity
was not due to the subnuclear location of enzymes which activate to
B(a)PDEI (Obi et al., 1986).
Primary cultures of epithelial and fibroblast cells derived
from human oral mucosa were studied for the ability to activate
B(a)P. The cells were exposed to B(a)P for 18 hours. B(a)P tetrols
and diols were the major metabolites formed by primary cultures of
epithelial and fibroblast cells derived from human oral mucosa. The
epithelial cells had a much higher rate of biotransformation of
B(a)P as measured by binding to cellular DNA. The major B(a)P-DNA
adduct was formed by the reaction of B(a)PDEI with the exocyclic 2-
amino group in guanine. In contrast to human cells, B(a)P phenols
and B(a)P 9,10-diol were the major metabolites produced by primary
epithelial and fibroblast cells derived from rat buccal mucosa. The
DNA binding levels of B(a)P in the two rat cell types were
identical, and the binding level was several-fold lower than in the
human epithelial cells (Autrup et al., 1985).
In vitro activation of B(a)P to protein-binding forms in high
yield was obtained with human and rat blood cells. A simple
combination of unsaturated fatty acid, i.e., linoleic or arachidonic
acid, and haematin or haemin resulted in activation of B(a)P
(Nemota, 1986).
Normal human mammary epithelial cell cultures and the human
mammary carcinoma T47D cell line were exposed to 3H-B(a)P for 24
hours, and the levels of binding were 81 and 182 pmol B(a)P/mg DNA
in normal and T47D cultures, respectively. Analysis of B(a)P-
deoxyribonucleoside adducts demonstrated the presence of three
adducts in both cells: (+)-anti-B(a)PDE-dGuo (B(a)PDEI-dGuo), (-)-
anti-B(a)PDE-dGuo, and syn-B(a)PDE-dGuo. Thus evidence was provided
that (-)-anti-B(a)PDE is formed in cell systems and reacts with DNA
in cells to form (-)-anti-B(a)PDE-dGuo (Pruess-Schwartz et al.,
1986).
The major B(a)P adduct formed in human mammary epithelial cells
was identified as B(a)PDEI-Guo. This adduct was only formed at very
low levels in rat mammary epithelial cells. The rat cells contained
a large proportion of syn-B(a)PDE adducts, and other unidentified
B(a)P-DNA adducts (Moore et al., 1987).
Studies on the metabolism of B(a)P in randomly proliferating
and confluent cultures of human skin fibroblast cells suggested that
factors other than random modification of DNA by B(a)P might have a
significant role in the expression of a transformed phenotype and
that metabolism and transformation are not directly related.
Furthermore, confluent dense cultures with a heightened capability
for metabolism of B(a)P were more active in the detoxification of
B(a)P than in the production of the metabolites associated with
cellular transformation (Cunningham et al., 1989).
The formation of adducts of B(a)P metabolites on DNA was
investigated in endometrial tissue from humans, hamsters, mice, and
rats. B(a)PDEI was the predominant adduct identified in all the
species studied. The amount of B(a)PDEI bound to DNA from human
endometrium was approximately 3 times higher than to DNA from
hamster tissue. Among the three animal species examined, the level
of this adduct was highest in hamsters and lowest in rats (Kulkarni
et al., 1986).
Human colon and bronchus tissue explants were incubated with
3H-B(a)P. The total percentage of metabolism of B(a)P was 8-59% in
bronchus and 11-23% in colon. B(a)P was found to bind covalently to
the DNA of human bronchi from 15 cases at a mean of 42 pmol/10 mg
DNA, and to the DNA of human colon from 6 cases at a mean of 66.5
pmol/10 mg DNA. The range among individuals was within one order of
magnitude. Human bronchus explant DNA contained one adduct: (+/-)-
B(a)PDEI-dGuo. DNA obtained from the lung or liver of rats given
2.0 mg/kg doses of 3H-B(a)P by intraperitoneal injection contained
3 DNA adducts in liver and two were observed in lung DNA
hydrolysates. One adduct from each organ cochromatographed with
(+/-)-B(a)PDEI-dGuo; however, the major adduct in each case eluted
earlier (Garner et al., 1985).
In vivo
The administration of B(a)P topically to pregnant C3H mice
during days 13-17 of gestation resulted in adduct formation in the
haemoglobin of the mother and progeny. Exposure to a total maternal
body burden of 500 µg B(a)P during the last 5 days of delivery
resulted in an average level of 6.35 pg of anti-diolepoxide
metabolite covalently attached per mg of haemoglobin analyzed in the
mother and 1.40 pg in the newborn animals. Concomitant adduct
formation in the DNA of the skin with B(a)P in the progeny was not
observed (Shugart & Matsunami, 1985).
Occurrence and persistence of DNA damage in the hepatic and
pulmonary tissues of fetal (days 12, 15 and 18 of pregnancy),
newborn (days 1 and 7) and adult (days 82-85) CD1 mice exposed to
selected doses of B(a)P (10 mg/kg body weight) were studied by
utilizing the alkaline elution technique. This approach indicated
that 15-day-old fetuses were the most sensitive to B(a)P
genotoxicity. B(a)P at dose levels of 0, 2 and 10 mg/kg body weight
was injected intraperitoneally into pregnant females or directly
into single fetuses and the fetal livers and lungs recovered 2, 4,
24 and 48 hours later. The results showed that the maximum DNA
damage is present at 4 hours following B(a)P treatment and it almost
disappeared at 48 hours irrespective of the route of B(a)P
administration. The effects where markedly magnified by Aroclor
pretreatment (Bolognesi et al., 1985).
In female ICR mice pregnancy lowered the binding of B(a)PDEI to
liver and lung DNA by 29-41%, but not the binding of other
metabolites (Lu et al., 1986a).
Using the 32P-postlabeling method the binding of B(a)P (200
µmol/kg body weight) to the DNA of various maternal and fetal
tissues was determined. B(a)P was administered to pregnant ICR mice
on day 18 of gestation. B(a)P was bound to the DNA of maternal and
fetal liver, lung, kidney, heart, brain, intestine, skin, maternal
uterus, and placenta, with organ-specific quantitative and
qualitative differences. B(a)P exhibited no obvious tissue
preference in either maternal or fetal organs. The fetal adduct
levels were generally lower than the corresponding maternal adduct
levels (Lu et al., 1986b).
The diolepoxide metabolite of B(a)P could be detected bound
covalently to the haemoglobin of erythrocytes isolated from mice
previously exposed to 400 µg B(a)P given intraperitoneally (Shugart,
1986).
Following a single oral administration of 80 umol/kg of B(a)P
to male BALB/c mice, a 32P-postlabeling assay showed that after 24
hours the highest levels of total DNA adducts were found in the
skin, followed by lung, liver and kidney. The main adduct
identified was B(a)PDEI-deoxy-guanosine 3',5'-bisphosphate (Schurdak
& Randerath, 1989).
The distribution and macromolecular binding of 3H-B(a)P was
examined in the skin, liver, lung, and stomach of SENCAR and BALB/c
mice following topical or oral administration of B(a)P (50 mg/kg
body weight) at time periods ranging from 0.5 to 48 hours. Levels
of labeled material in skin were higher, and the binding of B(a)P to
epidermal DNA was greater following topical administration than
following oral administration for mice of both strains. Following
oral administration of 3H-B(a)P greater levels of radioactivity
were found in liver, lung, and stomach tissue than after topical
administration (Morse & Carlson, 1985).
It has been reported that B(a)P is able to produce papillomas
of the skin in male SENCAR mice after a single oral administration
of 10 or 30 mg/kg body weight. When 3H-B(a)P was administered as
single doses orally or topically to male SENCAR mice, high
concentrations were found in the skin following topical application,
but very little material reached this target organ following oral
administration. In contrast, the internal organs generally
contained more material after oral administration. The binding of
labelled compound to DNA, RNA, and protein generally reflected the
distribution data, thus more compound was bound in the stomach,
liver, and lung after oral administration compared to topical
application, whereas the opposite was true for the skin (Carlson et
al., 1986).
The in vivo formation of B(a)P metabolite-DNA adducts has
been characterized in a variety of target and nontarget tissues of
A/HeJ mice and rabbits. Tissues included were lung, liver,
forestomach, colon, kidney, muscle, and brain. The major adduct
identified in each tissue was the (+)-(anti)-B(a)PDEI-dGuo adduct.
A (+/-)-syn-B(a)PDEII-dGuo adduct, a (-)-(anti)-B(a)PDEI-dGuo
adduct, and an unidentified adduct were also observed. The adduct
levels were unexpectedly similar in all the tissues examined from
the same B(a)P-treated animal. In mice given perorally 11.9 µg/kg
body weight the range was 12-28 fmol/µg DNA whereas in mice given
1190 µg/kg body weight the range was 2.7-6.1 pmol/µg DNA. For
example, the B(a)PDEI-DNA adduct levels in muscle and brain of mice
were approximately 50% of those in lung and liver at each oral B(a)P
dose used. Adduct levels formed in vivo in several cell types of
lung and liver were also examined. Macrophages, type II cells, and
Clara cells from lung and hepatocytes and non-parenchymal cells from
liver were isolated from B(a)P-treated rabbits. B(a)PDEI-
deoxyguanosine adduct was observed in each cell type and, moreover,
the levels were similar in various cell types. (Stowers & Anderson,
1985).
B(a)P-DNA adducts were analyzed in hepatic and pulmonary cells
isolated from rabbits 24 hours after intravenous administration of
3H-B(a)P (1 mg/kg). The major adduct in each of the cell types
analyzed was (+)-anti-B(a)PDEI-dGuo, but (+/-)-syn-B(a)PDEII-dGuo
and very low levels of (-)-anti-B(a)PDEI-dGuo and an unidentified
adduct were also observed. The level of the major adduct was
similar in each of the isolated cell types and was at least as high
in cells with very low cytochrome P-450-dependent monooxygenase
activity (hepatic nonparenchymal cells and alveolar macrophages) as
in those with higher activity (hepatocytes, alveolar type II cells,
and Clara cells) (Horton et al., 1985).
The 7-B(a)PDE-Gua adduct was identified in urine from partially
hepatectomized male Wistar rats treated intraperitoneally with 0,
10, 50 or 100 µg 3H-B(a)P in the urine. Less than 0.6% to 0.15% of
the doses were excreted as 7-B(a)PDE-dGua. In vitro studies using
human PLC/5 cells showed that the 7-BPDE-dGua adduct is very labile
and is released to the medium with a half life of 3 hours leaving
apurinic sites in the DNA (Autrup & Seremet, 1986).
The administration of the 3H-B(a)PDEI-DNA adduct, whether by
intraperitoneal or intravenous injection, to male Wistar rats
resulted in the majority of the radioactivity being recovered in the
faeces. Excretion was rapid: within 24 hours post-injection, 45% of
the applied dose was recovered in the faeces. HPLC analysis of
radioactive material extracted from the faeces by methanol showed
that it contained a single component which co-chromatographed with
3H-B(a)PDEI-dGuo (Tierney et al., 1987).
Animals dosed for 7 days with retinyl acetate (80 mg/kg body
weight/day), 13-cis-retinoic acid (13cisRA) (120 mg/kg body
weight/day), and N-(4-hydroxyphenyl)-retinamide (4HPR) (600 mg/kg
body weight/day), and showed a 38, 27, and 40% reduction in binding
of 3H-B(a)P (2 mg/kg body weight given intraperitoneally) to liver
DNA and a 29, 32, and 21% reduction in binding to stomach DNA,
respectively, when B(a)P was administered on the eighth day, and the
tissues were harvested 24 hours later. Binding to lung DNA was
reduced by 23 and 11%, respectively, in the 13cisRA- and 4HPR-dosed
rats. No differences were observed in binding to kidney (McCarthy
et al., 1987).
Following a single intraperitoneal injection of 3H-B(a)P, more
B(a)P was bound to liver DNA recovered from rats fed a diet
containing 20% menhaden fish oil (rich in omega-3 fatty acids) for
11 days at all time intervals tested (16, 24, 48, and 192 hours)
than was found from rats fed 0.5% menhaden oil. The increased
binding of 3H-B(a)P to liver DNA of rats fed the high level of
menhaden oil may be due, in part, to increases in the MFO
responsible for B(a)P activation (as suggested by increased
cytochrome P-450 level and total B(a)P hydroxylase activity)
(Dharwadkar & Wade, 1987).
Groups of 10 weanling male Wistar rats were subjected to
different levels of food restriction (0, 20, 40, and 60%
restriction). After 20 weeks on the diets there was significant
increase in the binding of 3H-B(a)P to hepatic DNA in 40 and 60%
food restricted animals (in vivo experimention), although this was
not observed under in vitro conditions. There was a decrease in
binding to pulmonary DNA and no change for renal DNA (Jagadeesan &
Krishnaswamy, 1989).
Blocking of in vivo arachidonic acid dependent prostaglandin
endoperoxide synthetase with acetylsalicylic acid (200 mg/kg body
weight) did not affect the in vivo activation of B(a)P to
metabolites capable of interacting irreversibly with cellular
macromolecules in guinea pig liver, lung, kidney, spleen, small
intestine, colon, and brain (Garattini et al., 1984).
2.2.7 Special studies on immunotoxicity
Young (3-6 months), middle-aged (16-18 months) and aged (23-26
months) mice were exposed in vitro and in vivo to B(a)P. The
generation of cells producing antibody to the T-dependent antigens
of sheep erythrocytes was observed to be suppressed in all age
groups. Significantly, aged mice were shown to exhibit a greater
percent suppression of antibody responses than young or middle-aged
mice both in vitro and in vivo (Lyte & Bick, 1985).
When B(a)P was incorporated into a T-dependent antibody (TDAb)-
producing spleen cell culture system, dose- and time-dependent
inhibition of plaque-forming cell responses was observed. Addition
of B(a)P at concentrations as low as 0.002 µg/ml resulted in
suppression of the TDAb plaque-forming cell response. In vitro
incorporation of B(a)P into polyclonal antibody-generating cultures
also resulted in a dose-related inhibition. Fourteen-day exposure
of mice to B(a)P (40 mg/kg body weight/day) resulted in 98%
suppression of the TDAb response. These studies suggest that the
suppressive effects of B(a)P are multicellular in origin, occur
apart from the carcinogenic effects, and cannot be attributed merely
to cellular toxicity (Blanton et al., 1986).
Progeny from B(a)P exposed (150 µg/g body weight) primiparous
mothers, injected during the second trimester of pregnancy, were
severely compromised immunologically. After 12-18 months the
progeny developed high incidences of hepatomas, lung adenomas and
adenocarcinomas, reproductive tumours, and lymphoreticular tumours
(Urso & Gengozian, 1980). When B(a)P was administered postnatally
(after 1 week) both immune suppression and tumour incidence was
substantially lower (Urso & Gengozian, 1982).
Pregnant C3H/Anf mice were exposed to 150 µg B(a)P/gram body
weight during fetogenesis (day 11-17 of gestation) and the progeny
were assayed for humoral and cell mediated immune responses at
different time intervals after birth. Immature offspring (1-4 wk)
were severely suppressed in their ability to produce antibody-
(plaque-) forming cells (PFC) against sheep red blood cells (SRBC)
and in the ability of their lymphocytes to undergo a mixed
lymphocyte response (MLR). A severe and sustained suppression in
the MLR and the PFC response occurred from the fifth month up to 18
months. Tumour incidence in the B(a)P-exposed progeny was 8- to 10-
fold higher than in those encountering corn oil alone from 18 to 24
months of age (Urso & Gengozian, 1984). Immunodeficiency
(abnormalities in the T cell-mediated responses caused by disruption
of T cell differentiation) occur early after birth (1 week) and
persists for 18 months (Urso & Johnson, 1987).
Pregnant C3H/Anfcum mice injected i.p. with 150 µg B(a)P/g body
weight at day 12. Within 5 days after injection, a 2- to 4-fold
reduction in leukocytes was observed when compared to controls which
persisted into the 10th postpartum day. The erythrocytes were also
significantly reduced but not to the same degree (1.2- to 1.5-fold).
Depression in white blood cells was attributed to lymphocyte
depletion (Urso et al., 1988). In the thymus, there was an
exacerbated depression in the amounts of thymocytes during pregnancy
relative to the controls, which was sustained postpartum. In the
spleen changes in the differentiation potential of T precursor cells
were indicated (Urso & Johnson, 1988).
The role of B(a)P metabolism in the suppression of the in
vitro humoral immune response was determined as the inhibition of
antibody-forming cells (AFC) of splenocyte cultures. Addition of
B(a)P or various B(a)P-diols in combination with addition of the
cytochrome P-450 inhibitor, alpha-naphthoflavone suggested that the
B(a)P-induced suppression of the in vitro AFC response is mediated
by B(a)P metabolites generated by cytochrome P-450 present within
splenocytes (Kawabata & White, 1987).
B(a)P administration (200 mg/kg body weight, i.p.) to female
B6C3F1 mice resulted in suppression of polyclonal responses and
substantial DNA adduct formation in mouse splenic keukocytes (SPL).
SPL adduct levels were similar to those in liver, lung, kidney, and
stomach. In vitro studies showed that SPL exhibited low AHH
activity and ability to form DNA-adducting metabolites, and that
rapid and dose related DNA adduct formation in SPL required the
addition of liver S9 (Ginsberg et al., 1989).
2.2.8 Special studies on bone marrow toxicity
Severe reduction in peripheral blood leucocytes was seen after
daily oral administration of B(a)P (120 mg/kg body weight/day) for
10-50 days in non-responsive mice (DBA/2), while only a mild effect
was seen in responsive mice (C57B1xDBA/2 F1). The responsive mice
were protected from bone marrow toxicity by marked induction of
B(a)P metabolism in the gastro-intestinal tract and liver (Nebert
et al., 1980).
Severe bone marrow depression with almost complete destruction
of pluripotent haematopoietic stem cells was seen in non-responsive
female DBA/2 mice after oral B(a)P (125 mg/kg body weight/day) for
13 days. Extreme resistance to bone marrow toxicity was observed in
responsive BDF1 mice fed for 19 days (Anselstetter & Heimpel, 1986).
The inducibility of P-4501 in the inbred mouse strains C57BL/6N
in the liver and intestine afforded protection against the
myelotoxic effect on bone marrow induced by B(a)P. In the DBA/2N
mouse strain having a poor-affinity Ah receptor, 120 mg/kg body
weight/day of B(a)P led to death within 3 weeks due to bone marrow
toxicity. B(a)P in the growth medium, in direct contact with
cultured myeloid cells, was more toxic to C57BL/6N than DBA/2N
cultured cells. Oral B(a)P induced P-4501 (measured by B(a)P-7,8-
dihydrodiol formation) in C57BL/6N but not DBA/2N intestine and
liver. In the bone marrow of oral B(a)P-treated C57BL/6N and DBA/2N
mice, the magnitude of P-4501 induction was about the same. Mice
having the high-affinity receptor, and therefore the P-4501
induction process in the intestine and liver, were protected from
oral B(a)P-induced myelotoxicity (Legraverand et al., 1983).
2.2.9 Special studies on atherosclerosis
Treatment of chickens for up to 20 weeks with weekly injections
of B(a)P (0.1, 1.0, and 10 mg/kg body weight/week) resulted in
significant increases in incidence and size of atherosclerotic
lesions of the abdominal aorta at the two higher doses used.
Administration of a single dose of B(a)P followed by the tumour
promoter TPA for 20 weeks did not have any effect (Bond et al.,
1981).
Weekly B(a)P injections of 0.1, 10 or 100 mg/kg body weight
were given to White Carneau Pigeons for 6 months. Only the high
dose treatment induced atherosclerosis in the aorta of the pigeons
(Revis et al., 1984).
The incidence of atherosclerotic lesions of aorta was increased
in mice given 0.15 mg 3-methylcholanthrene/kg body weight and
thereafter maintained on an atherogenic diet for 14 weeks. The
increase was significantly greater in responsive mice than in non-
responsive mice (Paigen et al., 1986).
2.3 Observations in humans
Polyclonal and monoclonal antibodies against B(a)PDEI-DNA
adducts have been developed and used in radioimmunoassays or
competitive ELISA assays in order to monitor human exposure to B(a)P
(Perera et al., 1982; Santella et al., 1984; 1985). The
monoclonal antibodies developed are not specific for B(a)PDEI-
adducts but also measure adducts from other PAHs (Santella, 1988).
The human monitoring studies have primarily been conducted on
occupationally exposed individuals and smokers.
B(a)P-DNA adducts were detected at low levels (0.08-0.16
fmol/µg DNA) in lung DNA from five of 27 patients with lung cancer
(Perera et al., 1982).
B(a)PDE-DNA adducts were detected in white blood cells from 7
of 28 roofers and 7 of 20 foundry workers (range 0.04 to 2.4 fmol/µg
DNA), and in two of 9 samples from non-occupationally exposed
volunteers (Shamsuddin et al., 1985).
Among the DNA samples from peripheral blood lymphocytes from 30
aluminium plant workers only one sample was found to contain a peak
similar to B(a)PDEI-DNA when measured by synchronous fluorescence
spectrophotometry (Vahakangas et al., 1985).
In coke oven workers exposed to high levels of PAHs, B(a)PDEI-
DNA adducts were detected in peripheral blood lymphoctyes from 18 of
27 individuals. In 9 controls no adducts were detected (Harris et
al., 1985).
A mean level of 1.7 fmol B(a)PDEI-DNA adduct/µg DNA measured by
radioimmunoassay was seen in 13 of 38 lymphocyte DNA samples from
coke oven workers exposed to high levels of PAHs. Four of the
samples were positive for B(a)PDEI-DNA in a synchronous fluorescence
spectrophotometry assay. These were also the samples having the
highest levels of DNA adducts (1.0 to >13.7 fmol/µg DNA) as
measured by radioimmunoassay (Haugen et al., 1986).
Blood monocytes from 45 selected patients with lung cancer and
30 healthy controls were incubated with 3H-B(a)P for 30 hours. The
DNA adducts were significantly elevated in 22 patients (non-smokers)
with early age cancer (4.34 fmol/micrograms of DNA). In 12 familial
cases (at least one first degree relative with lung cancer), a
slight elevation (2.77 fmol/micrograms of DNA) was not statistically
significant in comparison to healthy controls. B(a)P-DNA adduct
levels did not differ significantly between smokers and nonsmokers
(Rudiger et al., 1985).
DNA from placental specimens of smokers showed a small but not
statistically significant increase in B(a)PDEI-DNA adducts compared
to controls. The mean adduct levels were 1.65 and 0.96 fmol/µg DNA,
respectively. Using the 32P-postlabelling assay, an adduct not
derived from B(a)P was detected in 16 of 17 smokers compared to 3 of
14 nonsmokers (Everson et al., 1986).
In lymphocyte DNA of smokers and nonsmokers the number of
samples with detectable B(a)PDE-DNA adducts was much lower than in
placental DNA, but also did not differ between groups (Perera et
al., 1987).
Specimens of human lung, uterine cervix, ovary, and placenta
were studied for the presence of B(a)PDEI-DNA adducts by using
rabbit anti-B(a)PDE-DNA antibody and light microscopic
immunocytochemistry. B(a)PDEI-DNA antigenicity was detected in the
bronchial epithelial cells, cervical epithelium, oocytes, luteal
cells, corpora albicans, and hyalinized media of arteries within the
ovaries and trophoblastic cells of the placental villi (Shamsuddin &
Gan, 1988).
Breast epithelial cells from 10 donors were screened for the
existence of DNA adducts using the 32P-postlabeling assay. In
vitro studies had shown that the major B(a)P-DNA adduct formed by
human mammary epithelial cells in vitro was B(a)PDEI-dGuo. This
adduct did not appear to be formed by rat mammary cells exposed to
B(a)P in vitro. However, 32P-postlabeling analysis of mammary
epithelial cell DNA from rats exposed to B(a)P in vivo indicated
that B(a)PDEI-dGuo was a major B(a)P-DNA adduct under these exposure
conditions. When the mammary epithelial cells from 10 human donors
were screened for DNA adducts formed in situ, cells from three
donors exhibited distinct adduct patterns. None of these adducts
appeared to be B(a)PDEI-dGuo (Seidman et al., 1988).
In 40 of 81 lung cancer cases and 36 of 67 non cancer controls,
PAHs (B(a)PDEI)-DNA adducts in leukocytes were not significantly
related to age, sex, ethnicity, amount of cigarette smoking, dietary
charcoal, or caffeine consumption. The mean adduct level was about
0.4 fmol/µg DNA. When subjects were stratified by smoking status
(current, former, and nonsmoker), lung cancer cases who were current
smokers had significantly higher levels of adducts than current
smoker controls. A seasonal variation was observed, with peak in
adduct levels during July-October. The study indicates that
B(a)PDEI-DNA adducts reflect a pervasive and variable "background"
(Perera et al., 1989).
B(a)PDEI-dGuo and other apparent PAHs-DNA adducts were detected
in human peripheral lung tissue samples from 9 of 17 individuals
using HPLC-linked synchronous fluorescence spectrophotometry and
radioimmunoassay. No correlation between occupational or smoking
history was seen. A number of other (non-PAHs) adducts were also
detected (Wilson et al., 1989).
3. COMMENTS
Although benzo [a]pyrene was the substance on the agenda, the
Committee recognized that this was only one member of a class of
more than 100 compounds belonging to the family of polycyclic
aromatic hydrocarbons found in food and that they should be
considered as a class.
The Committee reviewed and discussed data from studies on the
toxicity of benzo [a]pyrene, with particular emphasis on its
toxicity after ingestion. The results demonstrated many different
toxic effects of this compound.
In mice, orally administered benzo [a]pyrene consistently
produced tumours of the forestomach and lung, and the few available
studies in rats showed tumours of the oesophagus, forestomach and
mammary gland. Tumours at other sites, such as lymphoreticular
tumours in mice, were also reported. The Committee noted that fetal
and newborn mice are especially vulnerable to the pulmonary and
lymphatic tumorogenicity of benzo [a]pyrene administered either by
direct injection or transplacentally.
The genotoxicity of benzo [a]pyrene both in vitro and in
vivo, is well documented, and this compound is consequently used
as a positive control substance in these types of studies. The
Committee noted that IARC had found inadequate evidence for
carcinogenicity of benzo [a]pyrene in humans, but sufficient
evidence for carcinogenicity to animals and for activity in short-
term genotoxicity tests.
The Committee also noted that in mice oral doses of 120 mg of
benzo [a]pyrene per kg of body weight and above caused intrauterine
toxic effects and fetal malformations when administered during
pregnancy.
The immunotoxicity and bone-marrow toxicity of benzo [a]pyrene
in mice were also considered. In the immunotoxicity studies, in
which pregnant mice received a single dose of benzo [a]pyrene at
150 mg per kg of body weight intrapreitoneally, the resultant
offspring were severley immunosuppressed. The Committee noted that
this effect may have led to the subsequent widespread development of
tumours in these animals.
Many studies have implicated benzo [a]pyrene-ne-7,8-diol-9,10-
oxide (BPDEI) as the proximate carcinogenic metabolite of
benzo [a]pyrene. This metabolite binds covalently to DNA, induces
mutations and transformations in short-term tests, and is a highly
potent carcinogen in mouse skin. However, the Committee noted that
studies in which benzo [a]pyrene was administered perorally to mice
and rabbits showed that the level of binding of BPDEI to DNA was
similar in all tissues examined (target tissues as well as non-
target tissues).
The Committee also considered studies in which the levels of
BPDEI-DNA adducts were measured in human tissues, although none of
these studies were aimed at monitoring benzo [a]pyrene exposure
from food. The Committee noted that the levels of DNA adducts were
elevated in only a few individuals, who were believed to have been
exposed to high levels of benzo [a]pyrene. The levels of BPDEI-DNA
adducts in humans were unrelated to factors such as age, sex,
ethnicity, number of cigarettes smoked, or caffeine consumption.
It was concluded that, for the purpose of evaluation, the most
significant toxicological effect of benzo [a]pyrene was its
carcinogenic activity.
The Committee had before it data from studies on
benzo [a]pyrene levels in various food and estimated dietary
intakes. These data demonstrated the wide-ranging levels of
benzo [a]pyrene in food and that these levels were dependent on
factors such as where the food was grown (i.e., industrialized or
non-industrialized area), how it was processed (e.g., smoking or
drying), and how it was cooked (e.g., charcoal grilling). In turn,
dietary intakes varied considerably, some consumers being exposed to
high levels of the substance.
The Committee noted that the estimated average daily intake of
benzo [a]pyrene by humans was about four orders of magnitude lower
than the level reported to be without effect on the incidence of
tumours in an experiment in rats in which benzo [a]pyrene was
incorporated in the diet. However, the Committee was unable to
establish a tolerable intake for benzo [a]pyrene, based on the
available data.
Nevertheless, the large difference between estimated human
intakes of benzo [a]pyrene and the doses producing tumours in
animals suggests that any effects on human health are likely to be
small. Despite this, the considerable uncertainties in risk
estimation require that efforts should be made to minimize human
exposure to benzo [a]pyrene as far as is practicable.
The Committee was informed that a long-term carcinogenicity
study in rats in which benzo [a]pyrene is being administered by
gavage has been initiated to investigate the dose-response
relationship for the tumorogenicity of this compound.
4. EVALUATION
The Committee acknowledged the complexities of the problem of
reducing exposure to B(a)P and other PAHs. Furthermore, it noted
that B(a)P exposure constitutes only a fraction of consumers'
exposure to PAHs and that some other members of this class of
compounds, not evaluated at this meeting, also exhibit a similar
toxicology profile to that of B(a)P and may thus contribute to the
overall carcinogenic risk. In this regard, strategies to minimize
B(a)P exposure would also be effective in reducing overall exposure
to PAHs. These include practices that consumers can effect, such as
cleaning fruits and vegetables thoroughly to remove any surface
contamination and, prior to barbecuing meats, trimming excess fat to
minimize "flare-ups" and cooking in a fashion that prevents contact
of the food with any flames. Measures that can be taken by the food
industry include conversion to indirect heating for drying foods,
switching to non-coal-fired roasters (e.g., for roasting coffee
beans), using protective coverings (e.g., cellulose casing) when
smoking foods conventionally, and ensuring compliance with limits
for PAHs in food additives specificed by national and or
international bodies. The Committee urged the application of these
measures to minimize contamination of food with polycyclic aromatic
hydrocarbons, including benzo [a]pyrene.
5. REFERENCES
ABE, S. (1986). Effects of arachidonic acid and indomethacin on
sister-chromatid exchange induction by polycyclic aromatic
hydrocarbons in mammalian cell lines. Mutat. Res., 173, 55-60.
ALINK, G.M., KUIPER, H.A., BEEMS, R.B. & KOEMAN, J.H. (1989). A
study on the carcinogenicity of human diets in rats: The influence
of heating and the addition of vegetables and fruit. Fd. Chem.
Toxicol., 22, 427-436.
ALZIEU, P. CASSAND, P., COLIN, C., GROILER, P. & NARBONNE, J.F.
(1987). Effect of vitamins A, C and glutathione on the mutagenicity
of benzo[a]pyrene mediated by S9 from vitamin A-deficient rats.
Mutat. Res., 192, 227-231.
ANDERSON, L.M., DESCHNER, E.E., ANGEL, M. & HERRMANN, S.L. (1982).
Murine colonic mucosal metabolism and cytotoxicity of
benzo[a]pyrene. Oncology, 39, 369-377.
ANDERSON, L.M., PRIEST, L.J., DESCHNER, E.E. & BUDINGER, J.M.
(1983). Carcinogenic effects of intracolonic benzo[a]pyrene in
beta-naphthoflavone-induced mice. Cancer Lett., 20, 117-123.
ANDERSON, L.M., ANGEL, M. & PRIEST, L.J. (1987). Lack of effect of
diet on benzpyrene metabolism by colonic mucosal homogenates from
mice. Arch. Toxicol., 60, 334-335.
ANSELSTETTER, V. & HEIMPEL, H. (1986). Acute haematotoxicity of
oral benzo(a)pyrene: the role of the Ah locus. Acta Haematol.
Basel, 76, 217-223.
ARCE, G.T., ALLEN, J.W., DOERR, C.L., ELMORE, E., HATCH, G.G.,
MOORE, M.M., SHARIEF, Y., GRUNBERGER, D. & NESNOW, S. (1987).
Relationships between benzo(a)pyrene-DNA adduct levels and genotoxic
effects in mammalian cells. Cancer Res., 47, 3388-3395.
AUTRUP, H., SEREMET, T., ARENHOLT, D., DRAGSTED, L. & JEPSEN, A.
(1985). Metabolism of benzo[a]pyrene-DNA cultured rat and human
buccal mucosa cells. Carcinogenesis, 6, 1761-1765.
AUTRUP, H. & SEREMET, T. (1986). Excretion of benzo[a]pyrene-Gua
adduct in the urine of benzo[a]pyrene treated rats. Chem. Biol.
Interactions, 60, 217-226.
AWOGI, T. & SATO, T. (1989). Micronucleus test with benzo[a]pyrene
using a single peroral administration and intraperitoneal injection
in males of the MS/Ae and CD-1 mouse strains. Mutat. Res., 223,
353-356.
BARBIERI, O., OGNIO, E., ROSSI, O., ASTIGIANO, S. & ROSSI, L.
(1986). Embryotoxicity of benzo[a]pyrene and some of its synthetic
derivatives in Swiss mice. Cancer Res., 46, 94-98.
BEEMS, R.B. (1984). Modifying effect of vitamin A on
benzo[a]pyrene-induced respiratory tract tumours in hamsters.
Carcinogenesis, 5, 1057-1060.
BEN-ITZHAK, J., LEVI, B.Z., SHOR, R., LANIR, A. BASSAN, H.M. &
ULITZUR, S. (1985). The formation of genotoxic metabolites of
benzo[a]pyrene by the isolated perfused rat liver, as detected by
the bioluminescence test. Mutat. Res., 147, 107-112.
BENJAMIN, H., STORKSON, J., TALLAS, P.G., & PARIZA, M.W. (1988).
Reduction of benzo[a]pyrene-induced forestomach neoplasms in mice
given nitrite and dietary soy sauce. Fd. Chem. Toxicol., 26, 671-
678.
BJORSETH, A. (1983). Sources and Emissions of PAHs In: Bjorseth, A
& Ramdahl, T. (eds), Handbook of Polycyclic Aromatic Hydrocarbons,
Marcel Dekker, Inc., New York and Basel, pp. 1-20.
BLANTON, R.H. LYTE, M., MYERS, M.J. & BLIC, P.H. (1986).
Immunomodulation by polyaromatic hydrocarbons in mice and murine
cells. Cancer Res., 46, 2735-2739.
BOCK, F.G., & DAO, T.L. (1961). Factors affecting the polynuclear
hydrocarbon level in rat mammary glands. Cancer Res., 21, 1024-
1029.
BOLOGNESI, C., ROSSI, L., BARBIERI, O. & SANTI, L. (1985).
Benzo[a]pyrene-induced DNA damage in mouse fetal tissues.
Carcinogenesis, 6, 1091-1095.
BOND, J. A., GOWN, A.M., YANG, H.L., BENDITT, E.P. & JUCHAU, M.R.
(1981). Further investigations of the capacity of polynuclear
aromatic hydrocarbons to elicit atherosclerotic lesions. J.
Toxicol. Environ. Health, 7, 327-335.
BONIN, A.M., STUPANS, I., BAKER, R.S., A.J. & HOLDER, G.M. (1985).
Effect of induction by DDT on metabolism and mutagenicity of
benzo[a]pyrene. Mutat. Res., 158, 111-117.
BOWES, S.G. & RENWICK, A.G. (1986a). The hepatic metabolism and
biliary excretion of benzo[a]pyrene in guinea-pigs fed normal, high-
fat or high-cholesterol diets. Xenobiotica, 16, 543-553.
BRUNE, H., DEUTSCH-WENZEL, R.P., HABS, M., IVANKOVIC, S. & SCHMÄL,
D. (1981). Investigation of the tumourigenic response to
benzo[a]pyrene in aqueous caffeine solution applied orally to
Sprague-Dawley rats. J. Cancer Res. Oncol., 102, 153-157.
BUENING, M.K., WISLOCKI, P.G., LEVIN, W., YAGI, H., THAKKER, D.R.,
AKAGI, H., KOREEDA, M., JERINA, D.M. & CONNEY, A.H. (1978).
Tumorigenicity of the optical enantiomers of the diasteriomeric
benzo[a]pyrene 7,8-diol-9,10-epoxides in newborn mice: Exceptional
activity of (+)-7ß,8a-dihydroxy-9a,10a-epoxy 7,8,9,10-
tetrahydrobenzo(a)-pyrene. Proc. Natl. Acad. Sci., 75, 5358-
5361.
BUI, Q.Q., TRAN, M.B. & WEST, W.L. (1986). A comparative study of
the reproductive effects of methadone and benzo[a]pyrene in the
pregnant and pseudopregnant rat. Toxicology, 42, 195-204.
BULAY, O.M. & WATTENBERG, L.W. (1970). Carcinogenic effect of
subcutaneous administration of benzo[a]pyrene during pregnancy on
the progeny. Proc. Soc. Exp. Bio. Med., 135, 84-86.
CARLSON, G.P., FOSSA, A.A., MORSE, M.A. & WEAVER, P.M. (1986).
Binding and distribution studies in the SENCAR mouse of compounds
demonstrating a route-dependent tumourigenic effect. Environ.
Health Perspect., 68, 53-60.
CHALMERS, J.G. & KIRBY, H. (1940). The elimination of 3:4-
benzpyrene from the animal body after subcutaneous injection. I.
Unchanged benzpyrene. Biochem., 34, 1191-1195.
CHU, E.W. & MALMGREN, R.A. (1965). An inhibitory effect of vitamin
A on the induction of tumours of forestomach and cervix in the
Syrian hamster by carcinogenic polycyclic hydrocarbons. Cancer
Res., 25, 884-895.
CLINTON, S.K. & VISEK, W.J. (1989). Wheat bran and the induction of
intestinal benzo(a)pyrene hydroxylase by dietary benzo[a]pyrene.
J. Nutr., 119, 395-402.
CONNEY, A.H. (1982). Induction of microsomal enzymes by foreign
chemicals and carcinogenesis by polycyclic aromatic hydrocarbons:
G.H.A. Clowes memorial lecture. Cancer Res., 42, 4875-4912.
COOPER, C.S. GROVER, P.L. & SIMS, P. (1983). The metabolism and
activation of benzo[a]pyrene. In: Bridges, J.W. & Chasseaud, L.F.
(eds.), Progress in Drug Metabolism, 7, John Wiley & Sons Ltd.,
Chichester, New York, Brisbane, Toronto, Singapore, pp. 295-396.
CUNNINGHAM, M.J., KURIAN, P. & MILO, G.E. (1989). Metabolism and
binding of benzo[a]pyrene in randomly-proliferating, confluent and
S-phase human skin fibroblasts. Cell. Biol. Toxicol., 5, 155-168.
DAHL, A.R., COSLETT, D.S., BOND, J.A. & HESSELTINE, G.R. (1985).
Metabolism of benzo[a]pyrene on the nasal mucosa of Syrian hamsters:
comparison to metabolism by other extrahepatic tissues and possible
role of nasally produced metabolites in carcinogenesis. J. Natl.
Cancer Inst., 75, 135-139.
DANHEISER, S.L., LIBER, H.L. & THILLY, W.G. (1989). Long-term, low-
dose benzo[a]pyrene-induced mutation in human lymphoblasts competent
in xenobiotic metabolism. Mutat. Res., 210, 143-147.
DAS, M., SETH, P.K. & MUKHTAR, H. (1985a). Distribution of
benzo[a]pyrene in discrete regions of rat brain. Bull. Environ.
Contam. Toxicol., 35, 500-504.
DAS, M., SRIVASTAVA, S.P., SETH, P.K. & MUKHTAR, H. (1985b). Brain
microsomal enzyme mediated covalent binding of benzo[a]pyrene to
DNA. Cancer Lett., 25, 343-350.
DENNIS, M.J., MASSEY, R.C., McWEENY, D.J. & KNOWLES, M.E. (1983).
Analysis of polycyclic aromatic hydrocarbons in UK total diets.
Fd. Chem. Toxicol., 21, 569-574.
DHARWADKAR, S.M. & WADE, A.E. (1987). B(a)P metabolism and the in
vivo binding to hepatic DNA of rats fed diets containing menhaden
fish oil. Nutr. Cancer., 10, 163-170.
DOCK, L., SCHEU, G., JERNSTROM, B., MARTINEZ, M., TORNDAL, U.B. &
ERIKSSON, L. (1988). Benzo[a]pyrene metabolism and induction of
enzyme-altered foci in regenerating rat liver. Chem. Biol.
Interact., 67, 243-253.
DONTENWILL, W. & MOHR, U. (1962). Experimentelle Untersuchungen zum
Problem der Carcinomentstehung im Respirationstrakt. I. Die
unterschiedliche Wirkung des Benzpyrens auf die Epithelien der
Mundhöhle und der Trachea des Goldhamsters. Z. Krebsforsch., 65,
56-61.
DOREMIRE, M.E., HARMON, G.E. & PRATT, D.E. (1979). A research note,
3,4-benzopyrene in charcoal grilled meats. J. Food Sci., 44, 622-
623.
EGESTAD, B., PETTERSSON, P., SJOVALL, J., RAFTER, J., HYVONEN, K. &
GUSTAFSSON, J.A. (1987). Studies on the chromatographic
fractionation of metabolites of benzo[a]pyrene in faeces and urine
from germfree and conventional rats. Biomed. Chromatogr., 2, 120-
134.
EVERSON, R.B., RANDERATH, E., SANTELLA, R.M., CEFALO, R.C., AVITTS,
T.A. & RANDERATH, K. (1986). Detection of smoking-related covalent
DNA adducts in human placenta. Science, 231, 54-57.
FAZIO, T. & HOWARD, J.W. (1983). Polycyclic Aromatic Hydrocarbons
in Foods. In: Bjorseth, A. (eds), Handbook of Polycyclic Aromatic
Hydrocarbons. Marcel Dekker, Inc., New York and Basel, pp. 461-717.
FOTH, H., KAHL, R. & KAH, G.F. (1988). Pharmacokinetics of low
doses of benzo[a]pyrene in the rat. Fd. Chem. Toxicol., 26, 45-
51.
FRETHEIM, K. (1983). Polycyclic aromatic hydrocarbons in grilled
meat products: a review. Food Chem., 10, 129-139.
FRITZ, W. (1971). Umfang und Quellen der Kontamination unserer
Lebensmittel mit Krebserzeugenden Kohlenwasserstoffen.
Ernärungsforchung, XVI, 547-557.
GARATTINI, E., COCCIA, P., ROMANO, M., JIRITANO, L.,NOSEDA, A. &
SALMONA, M. (1984). Prostaglandin endoperoxide synthetase and the
activation of benzo[a]pyrene to reactive metabolites in vivo in
guinea pigs. Cancer Res., 44, 5150-5155.
GARNER, R.C., STANTON, C.A., MARTIN, C.M., HARRIS, C.C. & GRAFSTROM,
R.C. (1985). Rat and human explant metabolism, binding studies, and
DNA adduct analysis of benzo[a]pyrene and its 6-nitro derivative.
Cancer Res., 45, 6225-6231.
GELBOIN, H.W. (1980). Benzo[a]pyrene metabolism, activation, and
carcinogenesis: role and regulation of mixed-function oxidases and
related enzymes. Physiol. Rev., 60, 1107-1166.
GIBEL, W. (1964). Experimenteller Beitrag zur Synkarzinogenese beim
Speiseröhrenkarzinom. Der Krebsarzt, 19, 268-272.
GINSBERG, G.L. & ATHERHOLT, T.B. (1989). Transport of DNA-adducting
metabolites in mouse serum following benzo[a]pyrene administration.
Carcinogenesis, 10, 673-679.
GINSBERG, G.L., ATHERHOLT, T.B. & BUTLER, G.H. (1989).
Benzo[a]pyrene-induced immunotoxicity: Comparison to DNA adduct
formation in vivo, in cultured splenocytes, and in microsomal
systems. J. Toxicol. Environ. Health, 28, 205-220.
GLATT, H. BUCKER, M., PLATT, K.L. & OESCH, F. (1985). Host-mediated
mutagenicity experiments with benzo[a]pyrene and two of its
metabolites. Mutat. Res., 156, 163-169.
GLATT, H., SEIDEL, A., RIBEIRO,O., KIRBY, C., HIROM, P. & OESCH, F.
(1987). Metabolic activation to a mutagen of 3-hydroxy-trans-7,8-
dihydroxy-7,8-dihydrobenzo[a]pyrene, a secondary metabolite of
benzo[a]pyrene. Carcinogenesis, 8, 1621-1627.
GOWER J.D. & WILLS, E.D. (1986). The dependence of the rate of
B(a)P metabolism in the rat small intestinal mucosa on the
composition of the dietary fat. Nutr. Cancer, 8, 151-161.
GRIMMER, G. (1979). Sources and Occurrence of Polycyclic aromatic
hydrocarbons. In: Egan, H., Castegharo, M., Kunte, H., Bogovski, P.,
& Walker, E.A. (eds)., Environmental Carcinogens. Selected Methods
of Analysis. IARC Publications No. 29, International Agency for
Research on Cancer, Lyon, 31-54.
GRIMMER, G. & POTT, F. (1983). Occurrence of PAHs. In: Grimmer, G.
(ed.), Environmental carcinogenesis: Polycyclic aromatic
hydrocarbons, CRC Press, Inc., Boca Raton, Florida, pp. 61-128.
GROVER, P.L. (1986). Pathway involved in the metabolism and
activation of polycyclic hydrocarbons. Xenobiotica, 16, 915-931.
HALL, M., FORRESTER, L.M., PARKER, D.K., GROVER, P.L. & WOLF, C.R.
(1989). Relative contribution of various forms of cytochrome P450
to the metabolism of benzo[a]pyrene by human liver microsomes.
Carcinogenesis, 10, 18515-1821.
HARRIS, C.C., VAHAKANGAS, K., NEWMAN, M.J., TRIVERS, G.E.,
SHAMSUDDIN, A., SINOPOLI, N., MANN, D.L. & WRIGHT, W.E. (1985).
Detection of benzo[a]pyrene diol epoxide-DNA adducts in peripheral
blood lymphocytes and antibodies to the adduct in serum from coke
oven workers. Proc. Natl. Acad. Sci. USA, 82, 6672-6676.
HAUGEN, A., BECHER, G., BENESTAD, C., VAHAKANGAS, K., TRIVERS, G.E.,
NEWMAN, M.J. & HARRIS, C.C. (1986). Determination of polycyclic
aromatic hydrocarbons in the urine, benzo[a]pyrene diol epoxide-DNA
adducts in lymphocyte DNA, and antibodies to the adducts in sera
from coke oven workers exposed to measured amounts of polycyclic
aromatic hydrocarbons in the work atmosphere. Cancer Res., 46,
4178-4183.
HECHT, S.S., GRABOWSKI, W. & GROTH, K. (1979). Analysis of faeces
for benzo[a]pyrene after consumption of charcoal-broiled beef by
rats and humans. Fd. Cosmet. Toxicol., 17, 223-227.
HEIDELBERGER, C. & WEISS, S.M. (1951). The distribution of
radioactivity in mice following administration of 3,4-benzpyrene-5-
14C and 1,2,5,6-dibenzanthracene-9,10-14C. Cancer Res., 11, 885-
891.
HORTON, J.K. ROSENIOR, J.C., BEND, J.R. & ANDERSON, M.W. (1985).
Quantitation of benzo(a)pyrene metabolite: DNA adducts in selected
hepatic and pulmonary cells types isolated from [3H]benzo[a]pyrene-
treated rabbits. Cancer Res., 45, 3477-3481.
HUGGINS, C. YANG, N.C. (1962). Induction and extinction of mammary
cancer. Science, 137, 257-262.
IANNACCONE, P.M., FAHL, W.E. & STOLS, L. (1984). Reproduction
toxicity associated with endometrial cell mediated metabolism of
benzo[a]pyrene: a combined in vitro, in vivo approach.
Carcinogenesis, 5, 1437-1442.
IARC (1973). IARC Monographs on the Evaluation of Carcinogenic Risk
of Chemicals to Man: Vol 3, Certain polycyclic aromatic
hydrocarbons and heterocyclic compounds. International Agency for
Research on Cancer, Lyon, 91-136.
IARC (1982). IARC Monographs on the Evaluation of Carcinogenic Risk
of Chemicals to Humans: Chemicals, industrial processes and
industries associated with cancer in humans, IARC monographs,
Volumes 1 to 29. Supplement 4, 227-228. International Agency for
Research on Cancer, Lyon.
IARC (1983). IARC Monographs on the Evaluation of Carcinogenic Risk
of Chemicals to Humans: Vol 32 Polynuclear aromatic compounds, Part
1, chemical, environmental and experimental data. International
Agency for Research on Cancer, Lyon, 33-91; 211-224.
JAGADEESAN, V. & KRISHNASWAMY, K. (1989). Effect of food
restriction on benzo[a]pyrene binding to DNA in Wistar rats.
Toxicology, 56, 223-226.
JONGENEELEN, F.J., LEIJDEKKERS, C.M. & HENDERSON, P.T. (1984).
Urinary excretion of 3-hydroxy-benzo[a]pyrene after percutaneous
penetration and oral absorption of benzo[a]pyrene in rats. Cancer
Lett., 25, 195-201.
JONGENEELEN, F.J., LEIJDEKKERS, C.M., BOX, R.P., THEUWS, J.L. &
HENDERSON, P.T. (1985). Excretion of 3-hydroxy-benzo[a]pyrene and
mutagenicity in rat urine after exposure to benzo[a]pyrene. J.
Appl. Toxicol., 5, 277-282.
KAPITULNIK, J., LEVIN, W., YAGI, H., & JERINA, D.M. (1977).
Benzo[a]pyrene 7,8-dihydrodiol is more carcinogenic than
benzo(a)pyrene in newborn mice. Nature, 266, 378-380.
KAPITULNIK, J., WISLOCKI, P.G., LEVIN, W., YAGI, H., JERINA, D.M. &
CONNEY, A.H. (1978). Tumourigenicity studies with diol-epoxides of
benzo(a)pyrene which indicate that (+/-)-trans-7ß,8alpha-dihydroxy-
9alpha,m,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is an
ultimate carcinogen in newborn mice. Cancer Res., 38, 354-358.
KATOH, Y., NEMOTO, N., TANAKA, M. & TAKAYAMA, S. (1983). Inhibition
of benzo[a]pyrene-induced mutagenesis in Chinese hamster V79 cells
by hemin and related compounds. Mutat Res., 121, 153-157.
KAWABATA, T.T. & WHITE, K.L., Jr. (1987). Suppression of the in
vitro humoral immune response of mouse splenocytes by benzo[a]pyrene
metabolites and inhibition of benzo[a]pyrene-induced
immunosuppression by alpha-naphthoflavone. Cancer Res., 47, 2317-
2322.
KLIGERMAN, A.D., EREXSON, G.L. & WILMER, J.L. (1985). Induction of
sister-chromatid exchange (SCE) and cell-cycle inhibition in mouse
peripheral blood B lymphocytes exposed to mutagenic carcinogens in
vivo. Mutat. Res., 157, 181-187.
KLIGERMAN, A.D., MOORE, M.M., EREXSON, G.L., BROCK, K.H., DOERR,
C.L., ALLEN, J.W. & NESNOW, S. (1986). Genotoxicity studies of
benz[1]aceanthrylene. Cancer Lett., 31, 123-131.
KLIGERMAN, A.D., NESNOW, S., EREXSON, G.L., EARLEY, K. & GUPTA, R.C.
(1989). Sensitivity of rat and mouse peripheral blood lymphocytes
to BaP adduction and SCE formation. Carcinogenesis, 10, 1041-
1045.
KOTIN, P., FALK, H.L. & BUSSER, R. (1959). Distribution, retention,
and elimination of C14-3,4-benzpyrene after administration to rats
and mice. J. Natl. Cancer Inst., 23, 541-555.
KULKARNI, M.S., CALLOWAY, K., IRIGARAY, M.F. & KAUFMAN, D.G. (1986).
Species differences in the formation of benzo[a]pyrene-DNA adducts
in rodent and human endometrium. Cancer Res., 46, 2888-2891.
LAHER, J.M., RIGLER, M.W., VETTER, R.D., BARROWMAN, J.A. & PATTON,
J.S. (1984). Similar bioavailability and lymphatic transport of
benzo[a]pyrene when administered to rats in different amounts of
dietary fat. J. Lipid Res., 25, 1337-1342.
LAM, L.K. & HASEGAWA, S. (1989). Inhibition of benzo[a]pyrene-
induced forestomach neoplasia in mice by citrus limonoids. Nutr.
Cancer, 12, 43-47.
LARSSON, B.K. (1982). Polycyclic aromatic hydrocarbons in smoked
fish. Z. Lebensm. Unters.-Forsch., 174, 101-107.
LARSSON, B.K., SAHLBERG, G.P., ERIKSSON, A.T. & BUSK, L.A. (1983).
Polycyclic aromatic hydrocarbons in grilled food. J. Agric. Food
Chem., 31, 867-873.
LARSSON, B.K. (1986). Polycyclic aromatic hydrocarbons in Swedish
foods. The National Food Administration, Sweden.
LAVOIE, E.J., STERN, S.L., CHOI, C.I., REINHARD, J. & ADAMS, J.D.
(1987). Transfer of the tobacco-specific carcinogens N'-
nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone and benzo[a]pyrene into the milk of lactating rats.
Carcinogenesis, 8, 433-437.
LESCA, P. (1983). Protective effects of ellagic acid and other
plant phenols on benzo[a]pyrene-induced neoplasia mice.
Carcinogenesis, 4, 1651-1653.
LEGRAVEREND, C., HARRISON, D.E., RUSCETTI, F.W. & NEBERT, D.W.
(1983). Bone marrow toxicity induced by oral benzo[a]pyrene:
protection resides at the level of the intestine and liver.
Toxicol. Appl. Pharmacol., 70, 390-401.
LEGRAVEREND, C., GUENTHNER, T.M. & NEBERT, D.W. (1984). Importance
of the route of administration for genetic differences in
benzo[a]pyrene-induced in utero toxicity and teratogenicity.
Teratology, 29, 35-47.
LEVIN, W., WOOD, A., CHANG, D.R., THOMAS, P., YAGI, H., THAKKER, D.,
VYAS, K., BOYD, C., CHU, S-Y., CONNEY, A. & JERINA, D. (1982).
Oxidative metabolism of polycyclic aromatic hydrocarbons to ultimate
carcinogens. Drug Metab. Rev., 13, 555-580.
LIJINSKY, W. & SHUBIK, P. (1964). Benzo[a]pyrene and other
polynuclear hydrocarbons in charcoal-broiled meat. Science, 145,
53-55.
LIJINSKY, W. & ROSS, A.E. (1967). Production of carcinogenic
polynuclear hydrocarbons in the cooking of food. Fd. Cosmet.
Toxicol., 5, 343-347.
LIOY, P.L., WALDMAN, J.M., GREENBERG, A., HARKOV, R. & PIETARINEN,
C. (1988). The Total Human Environmental Exposure Study (THEES) to
benzo[a]pyrene: comparison of the inhalation and food pathways.
Arch. Environ. Health, 43, 304-312.
LO, M-T. & SANDI, E. (1978). Polycyclic aromatic hydrocarbons
(polynuclears) in foods. Res Rev., 69, 35-86.
LU, L.J., DISHER, R.M. & RANDERATH, K. (1986a). Differences in the
covalent binding of benzo[a]pyrene, safrole, 1-hydroxysafrole, and
4-aminobiphenyl to DNA of pregnant and non-pregnant mice. Cancer
Lett., 31, 43-52.
LU, L.J., DISHER, R.M. REDDY, M.V. & RANDERATH, K. (1986b). 32p-
postlabeling assay in mice of transplacental DNA damage induced by
the environmental carcinogens safrole, 4-aminobiphenyl, and
benzo[a]pyrene. Cancer Res., 46, 3046-3054.
LUBET, R.A., NIMS, R.W., KISS, E., KOURI, R.E., PUTMAN, D.L. &
SCHECHTMAN, L.M. (1986). Influence of various parameters on
benzo[a]pyrene enhancement of adenovirus SA7 transformation of
Syrian hamster embryo cells. Environ. Mutagen., 8, 533-542.
LUTZ, W.K., VIVIANI, A. & SCHLATTER, C. (1978). Nonlinear dose-
response relationship for the binding of the carcinogen
benzo[a]pyrene to rat liver DNA in vivo. Cancer Res., 38, 575-
578.
LYTE, M. & BICK, P.H. (1985). Differential immunotoxic effects of
the environmental chemical benzo[a]pyrene in young and aged mice.
Mech. Ageing Dev., 30, 333-341.
MAHER, V.M., PATTON, J.D., YANG, J.L., WANG, Y.Y., YANG, L.L., AUST,
A.E., BHATTACHARYYA, N. & MCCORMICK, J.J. (1987). Mutations and
homologous recombination induced in mammalian cells by metabolites
of benzo[a]pyrene and 1-nitropyrene. Environ. Health Perspect.,
76, 33-39.
McCARTHY, D.J., LINDAMOOD, C.D. & HILL, D.L. (1987). Effects of
retinoids on metabolizing enzymes and on binding of benzo[a]pyrene
to rat tissue DNA. Cancer Res., 47, 5014-5020.
McNEILL, J.M. & WILLS, E.D. (1985). The formation of mutagenic
derivatives of benzo[a]pyrene metabolism in isolated perfused rat
lung and liver. Arch. Toxicol., 60, 270-277.
MOLLIERE, M., FOTH, H., KAHL, R. & KAHL, G.F. (1987). Comparison of
benzo[a]pyrene metabolism in isolated perfused rat lung and liver.
Arch. Toxicol., 60, 270-277.
MONTEITH, D.K., NOVOTNY, A., MICHALOPOULOS, G. & STROM, S.C. (1987).
Metabolism of benzo[a]pyrene in primary cultures of human
hepatocytes: dose-response over a four-log range. Carcinogenesis,
8, 983-988.
MOORE, C.J. & GOULD, M.N. (1984). Metabolism of benzo[a]pyrene by
cultured human hepatocytes from multiple donors. Carcinogenesis,
5, 1577-1582.
MOORE, C.J., PRUESS-SCHWARTZ, D., MAUTHE, R.J., GOULD, M.N. & BAIRD,
W.M. (1987). Interspecies differences in the major DNA adducts
formed from benzo[a]pyrene but not 7,12-dimethylbenz[a]anthracene in
rat and human mammary cell cultures. Cancer Res., 47, 4402-4406.
MORSE, M.A. & CARLSON, G.P. (1985). Distribution and macromolecular
binding of benzo[a]pyrene in SENCAR and Balb/c mice following
topical and oral administration. J. Toxicol. Environ. Health, 16,
263-276.
MUKHTAR, H., DEL-TITO, B.J., JR., MARCELO, C.L., DAS, M. & BICKERS,
D.R. (1984). Ellagic acid: a potent naturally occurring inhibitor
of benzo[a]pyrene metabolism and its subsequent glucuronidation,
sulfation and covalent binding to DNA in cultured BALB/C mouse
keratinocytes. Carcinogenesis, 5, 1565-1571.
MULLAART, E., BUYTENHEK, M., BROUWER, A., LOHMAN, P.H. & VIJG, J.
(1989). Genotoxic effects of intragastrically administered
benzo[a]pyrene in rat liver and intestinal cells. Carcinogenesis,
10, 393-395.
NEAL, J. & RIGDON, R.H. (1967). Gastric tumours in mice fed
benzo[a]pyrene. Tex. Rep. Biol. Med., 25, 553-557.
NEBERT, D.W. & JENSEN, N.M. (1979). Benzo[a]pyrene-initiated
leukemia in mice. Association with the allelic differences at the
Ah locus. Biochem Pharmacol., 27, 149-151.
NEBERT, D.W., JENSEN, N.M. LEVITT, R.C. & FLETON, J.S. (1980).
Toxic chemical depression of the bone marrow and possible aplastic
anemia explainable on a genetic basis. Clin. Toxicol., 16, 99-
122.
NEIS, J.M. & ROELOFS, H.M., VAN GEMERT, P.J. & HENDERSON , P.T.
(1986). Mutagenicity towards Salmonella typhimurium of some known
genotoxic agents, activated by isolated hepatocytes of monkey
(Macaca fascicularis). Comparison with isolated human
hepatocytes. Mutat. Res., 164, 139-143.
NEMOTO, N. (1986). Marked activation of benzo[a]pyrene to protein-
binding forms in the presence of unsaturated fatty acids and heme-
compounds. Carcinogenesis, 7, 267-271.
NEUBERT, D. & TAPKEN, S. (1988). Prenatal induction of
benzo(a)pyrene hydroxylases in mice. Arch. Toxicol., 62, 192-199.
OBI, F.O., RYAN, A.J. & BILLETT, M.A. (1986). Preferential binding
of the carcinogen benzo[a]pyrene to DNA in active chromatin and the
nuclear matrix. Carcinogenesis, 7, 907-913.
OCHI, T., ISHIGURO, T. & OHSAWA, M. (1985). The effects of cell
cycle position on the cytotoxicity and mutagenicity of
benzo[a]pyrene in cultured Chinese hamster cells. Toxicol. Lett.,
29, 183-190.
OESCH, F., BENTLEY, P., GOLAN, M. & STASIECKI, P. (1985).
Metabolism of benzo(a)pyrene by subcellular fractions of rat liver:
evidence for similar patterns of cytochrome P-450 in rough and
smooth endoplasmic reticulum but not in nuclei and plasma membrane.
Cancer Res., 45, 4838-4843.
O'NEILL, I.K., BINGHAM, S., POVEY, A.C., BROUET, I. & BEREZIAT, J.C.
(1990a). Modulating effects in human diets of dietary fibre and
beef, and time and dose on the reactive microcapsule trapping of
benzo[a]pyrene metabolites in the rat gastrointestinal tract.
Carcinogenesis, 11, 599-607.
O'NEILL, I.K., POVEY, A.C., BINGHAM, S. & CARDIS, E. (1990b).
Systematic modulation of human diet levels of dietary fibre and beef
on metabolism and disposition of benzo[a]pyrene in the
gastrointestinal tract of Fischer F344 rats. Carcinogenesis, 11,
609-616.
ORAVEC, C.T., JONES, C.A. & HUBERMAN, E. (1986). Activation of the
colon carcinogen 1,2-dimethylhydrazine in a rat colon cell-mediated
mutagenesis assay. Cancer Res., 46, 5068-5071.
PACIFICI, G.M., BLANCK, A. & RANE, A. (1986). Metabolism of
benzo[a]pyrene in the nuclear and microsomal fractions from rhesus
monkey liver. Acta Pharmacol. Toxicol., 568, 1-4.
PAIGEN, B., HOLMES, P.A., MORROW, A. & MITCHELL, D. (1986). Effect
of 3-methylcholanthrene on atherosclerosis in two congenic strains
of mice with different susceptibilities to methylcholanthrene-
induced tumors. Cancer Res., 46, 3321-3324.
PELKONEN, O. & NEBERT, D.W. (1982). Metabolism of polycyclic
aromatic hydrocarbons. Pharmacol. Rev., 34, 189-222.
PERERA, F.P., POIRIER, M.C., YUSPA, S.H., NAKAYAMA, J., JARETZKI,
A., CURNEN, M.M., KNOWLES, D.M. & WEINSTEIN, I.B. (1982). A pilot
project in molecular cancer epidemiology: determination of
benzo[a]pyrene-DNA adducts in animal and human tissues by
immunoassays. Carcinogenesis, 3, 1405-1410.
PERERA, F.P., SANTELLA, R.M., BRENNER, D., POIRIER, M.C., MUNSHI,
A.A., FISCHMAN, H.K. & Van RYZIN, J. (1987). DNA adducts, protein
adducts and sister chromatid exchange in lung cancer cases and
controls. J. Natl. Cancer Inst., 79, 449-456.
PERERA, F.P., MAYER, J., JARETZKI, A., HEARNE, S., BRENNER, D.,
YOUNG, T.L., FISCHMAN, H.K., GRIMES, M., GRANTHAM, S., TANG, M.X.,
TSAI, W.Y. & SANTELLA, R.M. (1989). Comparison of DNA adducts and
sister chromatid exchange in lung cancer cases and controls.
Cancer Res., 49, 4446-4451.
PEZZUTO, J.M., YANG, C.S., YANG, S.K., McCOURT, D.W. & GELBOIN, H.V.
(1978). Metabolism of benzo[a]pyrene and (-)-trans-7,8-dihydroxy-
7,8-dihydrobenzo[a]pyrene by rat liver nuclei and microsome.
Cancer Res., 38, 1241-1245.
PHILLIPS, D.H., HEWER, A. & GROVER, P.L. (1985). Aberrant
activation of benzo(a)pyrene in cultured rat mammary cells in vitro
and following direct application to rat mammary glands in vivo.
Cancer Res., 45, 4167-4174.
PHILLIPSON, C.E. & IOANNIDES, C. (1989). Metabolic activation of
polycyclic aromatic hydrocarbons to mutagens in the Ames test by
various animal species including man. Mutat. Res., 211, 147-151.
PIERCE, W.E.H. (1961). Tumour-promotion by lime oil in the mouse
forestomach. Nature, 189, 497-498.
PRASANNA, P., JACOBS, M.M. & YANG, S.K. (1987). Selenium inhibition
of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene
mutagenicity in Salmonella typhimurium strains TA98 and TA100.
Mutat. Res., 190, 101-105.
PRUESS-SCHWARTZ, D., BAIRD, W.M., NIKBAKHT, A., MERRICK, B.A. &
SELKIRK, J.K. (1986). Benzo[a]pyrene: DNA adduct formation in
normal human mammary epithelial cell cultures and the human mammary
carcinoma T47D cell line. Cancer Res., 46, 2697-2702.
RAHMAN, A., BARROWMAN, J.A. & RAHIMTULA, A. (1986). The influence
of bile on the bioavailability of polynuclear aromatic hydrocarbons
from the rat intestine. Can. J. Physiol. Pharmacol., 64, 1214-
1218.
RECIO, L. & HSIE, A.W. (1984). Glucuronide conjugation reduces the
cytotoxicity but not the mutagenicity of benzo[a]pyrene in the
CHO/HGPRT assay. Teratogen. Carcinog. Mutagen., 4, 391-402.
REES, E.D., MANDELSTAM, P., LOWRY, J. & LIPSCOMB, H. (1971). A
study of the mechanism of intestinal absorption of benzo[a]pyrene.
Biochem. Biophys. Acta, 225, 96-107.
REINERS, J.J., Jr. (1985). Differential effects of 7,8-benzoflavone
on polycyclic aromatic hydrocarbon dependent mutagenesis and
cytotoxicity in a keratinocyte cell-mediated mutation assay.
Cancer Lett., 26, 215-220.
REVIS, N.W., BULL, R., LAURIE, D., & SCHILLER, C.A. (1984). The
effectiveness of chemical carcinogens to induce atherosclerosis in
the White Carneau Pigeon. Toxicology, 32, 215-227.
RIBEIRO, O., KIRKBY, C.A., HIROM, P.C. & MILLBURN, P. (1985).
Secondary metabolites of benzo[a]pyrene: 3-hydroxy-trans-7,8-
dihydro-7,8-dihydroxybenzo[a]pyrene, a biliary metabolite of 3-
hydroxybenzo[a]pyrene in the rat. Carcinogenesis, 6, 1507-1511.
RIGDON, R.H. & NEAL, J. (1966). Gastric carcinomas and pulmonary
adenomas in mice fed benzo[a]pyrene. Tex. Rep. Biol. Med., 24,
195-207.
RIGDON, R.H. & NEAL J. (1969). Relationship of leukemia to lung and
stomach tumours in mice fed benzo[a]pyrene. Proc. Soc. Exp. Biol.,
130, 146-148.
RIVRUD, G.N. (1988). Mutagenicity testing of seminal fluid: seminal
fluid increases the mutagenicity of the precursor mutagen
benzo[a]pyrene in the presence of S9 mix. Mutat. Res., 208, 195-
200.
ROE, F.J.C. & WATERS, M.A. (1967). Induction of hepatoma in mice by
carcinogens of the polycyclic hydrocarbon type. Nature, 215, 299-
300.
ROSSI, L., BARBIERI, O., SANGUINETI, M., STACCIONE, A., SANTI, L.F.
& SANTI, L. (1983). Carcinogenic activity of benzo[a]pyrene and
some of its synthetic derivatives by direct injection into the mouse
fetus. Carcinogenesis, 4, 153-156.
RUDIGER, H.W., NOWAK, D., HARTMANN, K. & CERUTTI, P. (1985).
Enhanced formation of benzo[a]pyrene: DNA adducts in monocytes of
patients with a presumed predisposition to lung cancer. Cancer
Res., 45, 5890-5894.
RUDO, K.M., DAUTERMAN, W.C. & LANGENBACH, R. (1989). Human and rat
kidney cell metabolism of 2-acetylaminofluorene and benzo[a]pyrene.
Cancer Res., 49, 1187-1192.
RUGEN, P.L., STERN, C.D. & LAMM, S.H. (1989). Comparative
carcinogenicity of the PAHs as a basis for acceptable exposure
levels (AELs) in drinking water. Regulat. Toxicol. Pharmacol., 9,
273-283.
SAGAMI, I., OHMACHI, T., FUJII, H. & WATANABE, M. (1987).
Benzo[a]pyrene metabolism by purified cytochrome P-450 from 3-
methylcholanthrene-treated rats. Xenobiotica, 17, 189-198.
SALHAB, A.S., JAMES, M.O., WANG, S.L. & SHIVERICK, K.T. (1987).
Formation of benzo[a]pyrene-DNA adducts by microsomal enzymes:
comparison of maternal and fetal liver, fetal hematopoietic cells
and placenta. Chem. Biol. Interact., 61, 203-214.
SANTELLA, R.M., LIN, C.D., CLEVELAND, W.L. & WEINSTEIN, I.B. (1984).
Monoclonal antibodies to DNA modified by a benzo[a]pyrene diol
epoxide. Carcinogenesis, 5, 373-377.
SANTELLA, R.M., HSIEH, L.L., LIN, C.D., VIET, S. WEINSTEIN, I.B.
(1985). Quantitation of exposure to benzo[a]pyrene with monoclonal
antibodies. Environ. Health Perspect., 62, 95-99.
SANTELLA, R.M. (1988). Monitoring human exposure to carcinogens by
DNA adduct measurement. Cell Biol. Toxicol., 4, 511-516.
SANTODONATO, J., BASU, D. & HOWARD, P.F. (1980). Multimedia human
exposure and carcinogenic risk assessment for environmental PAHs.
In: Bjorseth, A. & Dennis, A.J. (eds.), Polynuclear aromatic
hydrocarbons: Chemistry and biological effects, Fourth International
Symposium, Battelle Press, Columbus, Ohio, pp. 435-454.
SANTODONATO, J., HOWARD, P. & BASU, D. (1981). Health and
ecological assessment of polynuclear aromatic hydrocarbons. J.
Environ. Pathol. Toxicol., 5, 1-364.
SCHAEFER, E.L., AU, W.W. & SELKIRK, J.K. (1985). Differential
induction of sister-chromatid exchanges by benzo[a]pyrene in variant
mouse hepatoma cells. Mutat. Res., 143, 69-74.
SCHAEFER-RIDDER, M., MOEROEY, T. & ENGELHARDT, U. (1984).
Inactivation of the thymidine kinase gene after in vitro
modification with benzo[a]pyrene-diol-epoxide and transfer to LTK-
cells as a eukaryotic test for carcinogens. Cancer Res., 44,
5861-5866.
SCHLEDE, E., KUNTZMAN, R., HABER, S. & CONNEY, A.H. (1970a). Effect
of enzyme induction on the metabolism and tissue distribution of
benzo[a]pyrene. Cancer Res., 30, 2893-2897.
SCHLEDE, E., KUNTZMAN, R. & CONNEY, A.H. (1970b). Stimulatory
effect of benzo[a]pyrene and phenobarbital pretreatment on the
biliary excretion of Benzo[a]pyrene metabolites in the rat. Cancer
Res., 30, 2898-2904.
SCHURDAK, M.E. & RANDERATH, K. (1989). Effects of route of
administration on tissue distribution of DNA adducts in mice:
comparison of 7H-dibenzo[c,g]carbazole, benzo[a]pyrene, and 2-
acetylaminofluorene. Cancer Res., 49, 2633-2638.
SEBTI, S.M., MOYNIHAN, C.G., ANDERSON, J.N. & BAIRD, W.M. (1985).
Benzo[a]pyrene-DNA adduct formation in target and activator cells in
a Wistar rat embryo cell-mediated V79 cell mutation assay.
Carcinogenesis, 6, 983-988.
SEIDMAN, L.A., MOORE, C.J. & GOULD, M.N. (1988). 32p-postlabeling
analysis of DNA adducts in human and rat mammary epithelial cells.
Carcinogenesis, 9, 1071-1077.
SHAMSUDDIN, A.K.M., SINOPOLI, N.T., HEMMINKI, K., BOESCH, R.R. &
HARRIS, C.C. (1985). Detection of benzo[a]pyrene: DNA adducts in
human white blood cells. Cancer Res., 45, 66-68.
SHAMSUDDIN, A.K. & GAN, R. (1988). Immunocytochemical localization
of benzo[a]pyrene-DNA adducts in human tissue. Hum. Pathol., 19,
309-315.
SHUGART, L. & MATSUNAMI, R. (1985). Adduct formation in hemoglobin
of the newborn mouse exposed in utero to benzo[a]pyrene.
Toxicology, 37, 241-245.
SHUGART, L. (1986). Quantifying adductive modification of
hemoglobin from mice exposed to benzo[a]pyrene. Anal. Biochem.,
152, 365-369.
SIEGFRIED, J.M., RUDO, K., BRYANT, B.J., ELLIS, S., MASS, M.J. &
NESNOW, S. (1986). Metabolism of benzo[a]pyrene in monolayer
cultures of human bronchial epithelial cells from a series of
donors. Cancer Res., 46, 4368-4371.
SIMS, P. & GROVER, P.L. (1974). Epoxides in polycyclic aromatic
hydrocarbon metabolism and carcinogenesis. Adv. Cancer Res., 20,
165-274.
SPARNINS, V.L., BARANY, G. & WATTENBERG, L.W. (1988). Effects of
organosulfur compounds from garlic and onions on benzo[a]pyrene-
induced neoplasia and glutathione S-transferase activity in the
mouse. Carcinogenesis, 9, 131-134.
SPINDLE, A. & WU, K. (1985). Developmental and cytogenetic effects
of caffeine on mouse blastocysts, alone or in combination with
benzo[a]pyrene. Teratology, 32, 213-218.
STOWERS, S.J. & ANDERSON, M.W. (1985). Formation and persistence of
benzo[a]pyrene metabolite-DNA adducts. Environ. Health Perspect.,
62, 31-39.
TEEL, R.W. (1984). A comparison of the effect of selenium on the
mutagenicity and metabolism of benzo[a]pyrene in rat and hamster
liver S9 activation systems Cancer Lett., 24, 281-289.
THAKKER, D.R., YAGI, H., AKAGI, H., KOREEDA, M., LU, A.Y.H., LEVIN,
W., WOOD, A.W., CONNEY, A.H. & JERINA, D.M. (1977). Metabolism of
benzo[a]pyrene. VI. Stereoselective metabolism of benzo[a]pyrene and
benzo[a]pyrene 7,8-dihydrodiol to diol epoxides. Chem.-Biol.
Interactions, 16, 281-300.
TIERNEY, B., MARTIN, C.N. & GARNER, R.C. (1987). Topical treatment
of mice with benzo[a]pyrene or parenteral administration of
benzo[a]pyrene diol epoxide-DNA to rats results in faecal excretion
of a putative benzo[a]pyrene diol epoxide-deoxyguanosine adduct.
Carcinogenesis, 8, 1189-1192.
T²TH, L. & BLAAS, W. (1973). Der Gehalt Gegrillter
Fleischerzeugnisse an Cancerogenen Kohlenwasserstoffen.
Fleischwirt., 53, 1456-1459.
T²TH, B. (1980). Tumorigenesis by benzo[a]pyrene administered
intracolonically. Oncology, 37, 77-82.
T²TH, L.& POTTHAST, K. (1984). Chemical aspects of the smoking of
meat and meat products. Adv. Food Res., 29, 87-158.
TRIOLO, A.J., APONTE, G.E. & HERR, D.L. (1977). Induction of aryl
hydrocarbon hydroxylase and forestomach tumors by benzo[a]pyrene.
Cancer Res., 37, 3018-3021.
URSO, P. & GENGOZIAN, N. (1980). Depressed humoral immunity and
increased tumour incidence in mice following in utero exposure to
benzo[a]pyrene. J. Toxicol. Environ. Health, 6, 569-576.
URSO, P. & GENGOZIAN, N. (1982). Alterations in humoral immune
response and tumour frequencies in mice exposed to benzo[a]pyrene
and X-rays before or after birth. J. Toxicol. Environ. Health,
10, 817-835.
URSO, P. & GENGOZIAN, N. (1984). Subnormal expression of cell-
mediated and humoral immune responses in progeny disposed toward a
high incidence of tumours after in utero exposure to
benzo[a]pyrene. J. Toxicol. Environ. Health, 14, 569-584.
URSO, P. & JOHNSON, R.A. (1987). Early changes in T lymphocytes and
subsets of mouse progeny defective as adults in controlling growth
of a syngeneic tumour after in utero insult with benzo[a]pyrene.
Immunopharmacology, 14, 1-10.
URSO, P. & JOHNSON, R.A. (1988). Quantitative and functional change
in T cells of primiparous mice following injection of benzo[a]pyrene
at the second trimester of pregnancy. Immunopharmacol.
Immunotoxicol., 10, 195-217.
URSO, P., RYAN, M.C. & BENNETT, J.S. (1988). Changes in peripheral
blood cells in mice after injection with benzo[a]pyrene during
pregnancy. Immunopharmacol. Immunotoxicol., 10, 179-93.
VAESSEN, H.A.M.G., SCHULLER, P.L., JEKEL, A.A. & WILBERS, A.A.M.M.
(1984). Polycyclic Aromatic Hydrocarbons in Selected Foods;
Analysis and Occurrence. Toxicol. Environ. Chem., 7, 297-324.
VAHAKANGAS, K., HAUGEN, A. & HARRIS, C.C. (1985). An applied
synchronous fluorescence spectrophotometric assay to study
benzo[a]pyrene-diolepoxide-DNA adducts. Carcinogenesis, 6, 1109-
1116.
Van CANTFORT, J., GIELEN, J.E. & NEBERT, D.W. (1985).
Benzo[a]pyrene metabolism in mouse liver. Association of both 7,8-
epoxidation and covalent binding of a metabolite of the 7,8-diol
with the Ah locus. Biochem. Pharmacol., 34, 1821-1826.
VESSELINOVITCH, S.D., KYRIASIS, A.P., MIHAILOVICH, N. & RAO, K.V.N.
(1975). Factors influencing augmentation and/or acceleration of
lymphoreticular tumours in mice by benzo[a]pyrene treatment.
Cancer Res., 35, 1963-1969.
WALTERS, J.M. & COMBES, R.D. (1986). Activation of benzo[a]pyrene
and aflatoxin B1 to mutagenic chemical species by microsomal
preparations from rat liver and small intestine in relation to
microsomal epoxide hydrolase. Mutagenesis, 1, 45-48.
WANG, C.X., WATANABE. K., WEISBURGER, J.H. & WILLIAMS, G.M. (1985).
Induction of colon cancer in inbred Syrian hamsters by intrarectal
administration of benzo[a]pyrene, 3-methylcholanthrene and N-methyl-
N-nitrosourea. Cancer Lett., 27, 309-314.
WARGOVICH, M.J., ENG. V.W. & NEWMARK, H.W. (1985). Inhibition by
plant phenols of benzo[a]pyrene-induced nuclear aberrations in
mammalian intestinal cells: a rapid in vivo assessment method.
Fd. Chem. Toxicol., 23, 47-49.
WARGOVICH, M.J. & ENG, V.W. (1989). Rapid screening of organosulfur
agents for potential chemopreventive activity using the murine NA
assay. Nutr. Cancer, 12, 189-193.
WATTENBERG, L.W. & LEONG, J.L. (1970). Inhibition of the
carcinogenic action of benzo[a]pyrene by flavones. Cancer Res.,
30, 1922-1925.
WATTENBERG, L.W. (1972). Inhibition of carcinogenic and toxic
effects of polycyclic hydrocarbons by phenolic antioxidants and
ethoxyquin. J. Natl. Cancer Inst., 48, 1425-1430.
WATTENBERG, L.W. (1973). Inhibition of chemical carcinogen-induced
pulmonary neoplasia by butylated hydroxyanisole. J. Natl. Cancer
Inst., 50, 1541-1544.
WATTENBERG, L.W. (1974). Inhibition of carcinogenic and toxic
effects of polycyclic hydrocarbons by several sulfur-containing
compounds. J. Natl. Cancer Inst., 52, 1583-1587.
WATTENBERG, L.W. (1977). Inhibition of carcinogenic effects of
polycyclic hydrocarbons by benzyl isothiocyanate and related
compounds. J. Natl. Cancer Inst., 58, 395-398.
WEI, C.E., ALLEN, K. & MISRA, H.P. (1989). Role of activated oxygen
species on the mutagenicity of benzo[a]pyrene. J. Appl. Toxicol.,
9, 169-173.
WELCH, R.M., GOMMI, B., ALVARES, A.P. & CONNEY, A.H. (1972). Effect
of enzyme induction on the metabolism of benzo[a]pyrene and 3-
methyl-4-monomethylaminoazobenzene in the pregnant and fetal rat.
Cancer Res., 32, 973-978.
WEST, C.E. & HORTON, B.J. (1976). Transfer of polycyclic
hydrocarbons from diet to milk in rats, rabbits and sheep. Life
Sciences, 19, 1543-1552.
WEST, C.E., ALLEN, K. & MISRA, H.P. (1989). Role of activated
oxygen species on the mutagenicity of benzo[a]pyrene. J. Appl.
Toxicol., 9, 169-173.
WEYAND, E.H. & BEVAN, D.R. (1986). Benzo[a]pyrene disposition and
metabolism in rats following intratracheal instillation. Cancer
Res., 46, 5655-5661.
WHO (1987). Air quality guidelines for Europe. World Health
Organization Regional Office for Europe, Copenhagen, WHO Regional
Publications, European Series No. 23, pp. 105-117.
WIERSMA, D.A., STEMMER, P.M. & ROTH, R.A. (1984). Influence of red
blood cells, serum albumin, and serum lipoproteins on the clearance
of benzo[alpha]pyrene by isolated livers of 3-methylcholanthrene-
treated rats. Biochem. Pharmacol,. 33, 3433-3438.
WILSON, N.M., CHRISTOU, M., TURNER, C.R., WRIGHTON, S.A. & JEFCOATE,
C.R. (1984). Binding and metabolism of benzo[a]pyrene and 7,12-
dimethyl-benz[a]anthracene by seven purified forms of cytochrome P-
450. Carcinogenesis, 5, 1475-1483.
WILSON, V.L. & JONES, P.A. (1984). Chemical carcinogen-mediated
decreases in DNA 5-methylcytosine content of BALB/3T3 cells.
Carcinogenesis, 5, 1027-1031.
WILSON, V.L., WESTON, A., MANCHESTER, D.K., TRIVERS, G.E., ROBERTS,
D.W., KADLUBAR, F.K., WILD, C.P., MONTESANO, R., WILLEY, J.C., MANN,
D.L. & HARRIS, C.C. (1989). Alkyl and aryl carcinogen adducts
detected in human peripheral lung. Carcinogenesis, 10, 2149-2153.
See Also:
Toxicological Abbreviations
BENZO[a]PYRENE (JECFA Evaluation)
Benzo[a]pyrene (IARC Summary & Evaluation, Volume 32, 1983)