BIS (2-ETHYLHEXYL)PHTHALATE
EXPLANATION
Bis (2-ethylhexyl)phthalate (DEHP) was evaluated previously at
the twenty-eighth meeting of the Joint FAO/WHO Expert Committee on
Food Additives (Annex 1, reference 66). The information evaluated
included results of pharmacokinetic and metabolism studies of DEHP,
data from reproduction, teratogenicity, mutagenicity, short- and
long-term toxicity, and carcinogenicity studies, and results of
special studies on testicular atrophy and on the relationship of
DEHP administration to peroxisome proliferation in the liver. At
that time it was concluded that DEHP is a hepatocarcinogen in both
rats and mice. The Committee recommended that human exposure to
DEHP as a result of its migration from food-contact materials be
reduced to the lowest level technologically attainable. The
Committee provisionally accepted the use of food-contact materials
that contain bis (2-ethylhexyl)phthalate as a potential migrant
into food, subject to the conditions outlined in Section 2.2 and
the first paragraph of Section 3.2 of that report.
Since the previous evaluation, additional data have become
available on mechanisms of testicular atrophy and DEHP-induced
hepatocarcinogenicity. Additional data relevant to these two
mechanisms are summarized and discussed in the following monograph
addendum.
BIOLOGICAL DATA
Special studies on testicular effects, fertility, and teratogenicity
Rats, mice, guinea pigs, and ferrets have been shown to be
susceptible to the induction of testicular injury by DEHP and other
phthalate esters, but Syrian hamsters are comparatively resistant
(Creasey et al., 1983; Gray et al., 1982b). In rats, repeated
administration of DEHP results in seminiferous tubular atrophy that
is characterized by a loss of the meiotic and post-meiotic germ
cell populations from the seminiferous epithelium (Creasey et
al., 1983), accessory sex gland atrophy, reduced testicular and
anterior prostate zinc concentrations (Curto & Thomas, 1982), and
increased testicular testosterone concentration (Oishi & Hiraga,
1980). Characteristic testicular changes are similar in all
susceptible species, involving early detachment of spermatocytes
and spermatids from the seminiferous epithelium (Gray & Gangolli,
1986). Morphological changes are evident in Sertoli cells within 3
to 6 hours following a single dose of di-n-pentyl phthalate, at
which time the germ cell population appeared unaffected (Creasey
et al., 1983).
Oishi & Hiraga (1983) demonstrated that testicular atrophy
induced by DEHP could not be prevented by co-administration of
zinc. The ability of dietary zinc and co-administered testosterone
to inhibit DEHP-induced testicular atrophy in rats also has been
studied. Agarwal et al. (1986) reported that adult male F344 rats
on a zinc-deficient diet showed an enhanced susceptibility to the
gonadotoxic effects of DEHP and argued that this finding supports
the hypothesis that testicular zinc depletion is casually related
to the ensuing testicular and accessory sex organ atrophies
produced by DEHP. Parmar et al. (1987) reported that co-
administration of testosterone and DEHP to adult male albino Wistar
rats appeared to prevent testicular injury induced by DEHP
administration alone. The authors suggested that these results
argue for the involvement of testosterone in DEHP-induced
testicular atrophy.
Effects of age and hormones on induction of testicular atrophy
in rats were investigated by Gray & Gangolli (1986). DEHP (0 and
2800 mg/kg/day) was administered orally as corn oil solutions to
six groups of eight male Sprague-Dawley rats (one group of four-,
10-, and 15-week old rats per dose). DEHP was administered for 10
days, after which the rats were killed. Administration of DEHP to
four-week old rats produced a marked depression in the weight of
the testes, seminal vesicle, and prostate. In 10-week old rats,
DEHP administration produced only a slight reduction in testis
weight but the weights of the seminal vesicle and prostate were
significantly reduced. In 15-week old rats, DEHP had no effect on
any of these organ weights. Histopathologically, the testes of the
treated four-week old rats showed severe atrophy affecting
virtually all tubules; tubules were populated only by Sertoli
cells, spermatogonia, and occasional primary spermatocytes. In the
10-week old treated rats, these histological changes were present in
from 5 to 50% of tubules; non-affected tubules appeared normal. No
histological abnormalities were seen in testes from treated 15-week
old rats.
The age-dependent response of the rat testes to DEHP was also
studied by Sjoberg et al. (1986b). Groups of male Sprague-Dawley
rats (25, 40, and 60 days old at the beginning of the experiment)
were given DEHP in the diet (dose adjusted to give a daily intake
of 0, 1.0, or 1.7 g/kg bw) for 14 days; rats were then killed.
Testicular weight was markedly reduced in the 25- and 40-day old
rats given 1.7 g/kg DEHP. All tubules were affected with severe
testicular damage, in the 25-day old rats. When the mean daily
intake of DEHP was 1.0 g/kg, however, only a few tubules in each of
the 25- and 40-day old animals was affected.
The possibility that DEHP induces testicular atrophy by
interfering with the production of testosterone or the pituitary
gonadotrophins was examined in studies involving co-administration
of these hormones with di-n-butyl phthalate (DBP). Six groups of
six male Sprague-Dawley rats (4 to 5 weeks old) were given DBP
orally as corn oil solutions (0 and 2000 mg/kg/day) for five days,
after which they received 50 units of pregnant mares' serum
gonadotrophin (PMSG) in corn oil by subcutaneous injection on the
first two days of DBP treatment, and one group of rats from each
dosage level received an aqueous solution of testosterone
propionate (200 µg/kg/day) by subcutaneous injection daily during
treatment with DBP. Rats treated with DBP alone showed a
significant reduction in testis and seminal vesicle weight and
severe testicular atrophy. Administration of PMSG or testosterone
propionate did not markedly influence the effects of DBP on the
testis. The authors concluded that these results suggest testicular
lesions caused by DBP and other phthalate esters are not primarily
due to lack of availability of pituitary hormones or testosterone,
thus pointing to a site of action in the seminiferous tubules (Gray
& Gangolli, 1986).
Gray & Gangolli (1986) also studied the effects of some
phthalate esters on two specific markers of Sertoli cell function,
the secretion of seminiferous tubule fluid and of androgen binding
protein (ABP), in 4- to 50 week old male phthalate in corn oil
(2200 mg/kg), a production of fluid and ABP was almost completely
suppressed. This effect was still marked at a dose of 440 mg/kg,
but was not evident at 220 mg/kg. After three daily doses of DBP at
220 mg/kg, however, one out of five rats was partially affected. A
single oral dose of mono-2-ethylhexyl phthalate in corn oil (MEHP;
1000 mg/kg), the principal metabolite of orally administered DEHP,
reduced fluid and ABP production to approximately 50% of control
levels. After three daily doses of MEHP (1000 mg/kg), fluid and ABP
production was approximately 25% of control levels. However,
diethyl phthalate, an ester that does not cause testicular atrophy
(Foster et al., 1980), had no effect on these criteria of Sertoli
cell function after tree daily oral doses of 1600 mg/kg in corn oil
(a dose level equimolar with 220 mg/kg DBP).
In contrast to the preceding results, when 10-week old male
Sprague-Dawley rats were given a single oral dose of DBP in corn
oil (2200 mg/kg), seminiferous tubule fluid and androgen binding
protein production were only reduced to approximately 60% of
control levels; oral administration of MEHP (1000 mg/kg in corn
oil) produced no effect. MEHP was still without effect after three
daily doses (Gray & Gangolli, 1986).
Several researchers have attempted to identify the active
metabolite of DEHP that affects the rat testis in vivo and
in vitro. Sjoberg et al. (1986a) reported that no testicular
damage was observed in young male Sprague-Dawley rats receiving
oral doses of DEHP or 2-EH (2.7 mmol/kg bw) daily for five days.
In animals receiving corresponding doses of MEHP, however, the
number of degenerated spermatocytes and spermatids was increased,
whereas no such effects were seen in animals given MEHP-derived
metabolites. MEHP (at concentrations as low as 10 Molar) was the
only compound that enhanced germ cell detachment from mixed primary
cultures of Sertoli and germ cells. The authors suggested that
these results indicate the probability that effects on the testes
observed after administration of DEHP are exerted by its metabolite
MEHP.
Because of the effects of phthalate esters on Sertoli cells
and the early separation of germ cells from Sertoli cells observed
in vivo, Gray & Beamand (1984) examined the use of primary
cultures of Sertoli and germ cells as an in vitro model for
phthalate-induced testicular toxicity. Addition of 100 µM MEHP to
the culture medium for 24 hours resulted in a pronounced detachment
of germ cells from the Sertoli cell monolayer and a change in
Sertoli cell morphology to a more elongated shape. The effect of
MEHP was shown to be concentration dependent over the range of 1 to
100 µM. No such changes were produced by DEHP (up to 100 µM) or by
its other primary metabolite, 2-ethylhexanol (EH). Three
metabolites of MEHP (compounds V, VI, and IX described by Albro et
al. in 1973) were also tested: compounds V and VI had no effect at
100 µM, but compound IX did produce a slight increase in germ cell
detachment. In studies with a range of phthalate monoesters, it was
found that only those causing testicular damage in vivo produced
an increase in germ cell detachment at low concentrations (1 to 100 µM)
in culture; of the phthalate monoesters tested, MEHP produced the most
marked response (Gray & Gangolli, 1986). Finally, because the germ
cells detaching from the monolayer cultures were viable and
morphologically normal, and because morphological changes were seen
in the still-attached Sertoli cells, Gray & Gangolli (1986) suggested
that these results indicate that the action of phthalates may be
mediated via a primary effect on Sertoli cells.
Saxena et al. (1985) investigated the role of several
enzymes in DEHP-induced testicular injury in rats. Two groups of
six male Wistar albino rats (13 weeks old) were given either saline
or DEHP (2000 mg/kg bw) orally for 7 consecutive days. All animals
were sacrificed on day 8 of the experiment. Following treatment
with DEHP, relative organ weight of the testis was not
significantly different from that of controls. Histopathology of
DEHP-treated testes, however, revealed focal interstitial edema and
degenerative changes in seminiferous tubules. The following enzyme
changes were observed following DEHP treatment: succinic
dehydrogenase activity, distributed throughout the seminiferous
tubules and interstitial tissue in control rats, was markedly
reduced; glucose-6-phosphate dehydrogenase activity, demonstrated
in interstitial cells and tubular epithelium of control animals,
was increased in interstitial tissue of the testes; NADH-diaphorase
activity, localized in seminiferous tubules and interstitial tissue
in control rats, demonstrated decreased activity in the
interstitial tissue where edema was not noted; ATPase activity,
observed in the basement membrane and interstitial tissue of
control rats, was enhanced; alkaline phosphatase activity,
localized in basement membrane and interstitial tissue of control
animals, was slightly enhanced; and acid phosphatase activity,
observed in the form of granules throughout the cross section of
the control rat, was reduced. The authors suggested that these
alterations indicate that disruption of cellular energetics in the
testes may be responsible for DEHP-associated infertility in male
rats.
Changes in cell-specific enzyme activities during DEHP-induced
testicular atrophy were also investigated by Oishi (1986). Ten
groups of seven male Wistar rats (30 days old) were administered
saline (2 ml/kg/day) or DEHP (2 g/kg/day, without vehicle) by
gavage daily. One pair each of the control and DEHP-treated groups
was sacrificed after 0, 1, 3, 6, or 10 days. Specific activities of
testicular enzymes associated with postmeiotic spermatogenic cells,
such as lactate dehydrogenase isozyme-X, hyaluronidase, and
sorbitol dehydrogenase, were lower than those of control animals by
day 10, which was coincident with degeneration of spermatogenic
cells in this experiment. The specific activities of enzymes
associated with premeiotic spermatogenic cells, Sertoli cells, or
interstitial cells were higher than those of control animals by day
10. The specific activities of alcohol dehydrogenase and aldolase,
zinc-containing enzymes, increased after DEHP treatment in spite of
the decrease of zinc concentration in the testis. The authors
noted, however, that all of these changes occurred after or
simultaneous with massive histological or morphological changes
rather than prior to such changes.
Parmar et al. (1986) observed increases in the activities of
gamma-glutamyl transpeptidase and lactate dehydrogenase in adult
male albino rats receiving oral doses of 250, 500, 1000 and 2000
mg/kg DEHP in groundnut oil per day for 15 days. An increase in the
activity of beta-glucuronidase and decrease in the activity of acid
phosphatase was observed at the highest dose of DEHP. The authors
concluded that these results suggest that DEHP can affect
spermatogenesis by altering the activities of enzymes responsible
for the maturation of sperm.
Agarwal et al. (1986) investigated the recovery from
DEHP-induced testicular toxicity produced upon discontinuance of
exposure in sexually mature rats. Five groups of 24 male F344 rats
(15-16 weeks old) were administered DEHP in the diet (0, 320, 1250,
5000, or 20,000 mg/kg diet; ppm) for 60 consecutive days. Eight
rats from each group were sacrificed after having been maintained
on a normal (not containing DEHP) diet for five days following the
60-day treatment period; the remaining sixteen rats per group were
placed on a normal diet for 70 days, than killed. Dietary
administration of DEHP produced toxicity evidenced by reduced
testicular and accessory organ weights, loss of testicular zinc,
and induction of seminiferous tubular atrophy. The toxic response
was dose-dependent and statistically significant with varying
severity depending upon the target tissue: Histopathological
evidence of tissue injury to the testis was characterized by severe
atrophy of the seminiferous tubules and loss of spermatogenesis in
rats fed a diet containing 20,000 ppm DEHP. Cessation of exposure
to DEHP initiated partial to complete recovery from toxicity in
most cases; magnitudes of recovery were available, with the gonads
being slower that other systems (such as liver).
Oishi (1985), however, found limited reversibility of
testicular atrophy induced by DEHP in young rats. Two groups of 20
male Wistar rats (95-112 g) were administered either a saline
solution (2 ml/kg/day) or DEHP (2.0 g/kg/day, without vehicle) by
gavage for 14 days. Ten rats from each group were killed one day after
treatment. The remaining ten animals per group were killed 45 days
after cessation of administration of saline or DEHP. For rats
killed on day 15 of the experiment, testicular and accessory sex
organ weights for rats administered DEHP were significantly less
than those of control animals. Histological changes in the testes
of DEHP-treated rats were characterized by a marked shrinkage of
seminiferous tubules: The germinal epithelium consisted of only
Sertoli cells, very few spermatogonia, and several multinucleated
cells. At 45 days after termination of DEHP administration, the
majority of tubules showed little more than a lining of Sertoli
cells, although a small number showed a partially intact germinal
epithelium. Spermatocytes, spermatids, and spermatozoa were seen in
the few tubules in which spermatogenesis was regenerated. The
percentage of spermatogenic tubules in a representative cross
section was 0 and 12.8%, respectively, at termination of the 2-week
DEHP treatment and following the 45-day recovery period.
Douglas et al. (1986) studied mutagenic and other genotoxic
effects of phthalate esters in adult mice and rats and in Chinese
hamster ovary cells in vitro. When 6-8 week old B6C3F1 mice (five
mice per group) were given intraperitoneal injections of DEHP in
olive oil (0, 0.6, 3.0, or 6.0 g/kg bw/day) for five consecutive days,
the numbers of morphologically abnormal sperm in treated groups did
not differ from controls in the 12 weeks following treatment. When
6-8 week old Sprague-Dawley rats (three rats per group) were given
intraperitoneal injections of DEHP in olive oil (0, 0.52, 2.6, or
5.2 g/kg bw/day) for five consecutive days, the numbers of
morphologically abnormal sperm were also unaffected by DEHP
treatment. In addition, DEHP treatment of Chinese hamster ovary
cultures at concentrations up to 10 mM for one hour induced neither
sister chromatid exchange nor DNA damage.
The antifertility and mutagenic effects of parenteral
administration of DEHP to mice was investigate by Agarwal et al.
(1985) using a modified dominant lethal test emphasizing the
repeated administration of small doses of the test compound. Adult
male and female ICR mice (8-10 weeks old) were used in the
experiment. Groups of eight male mice were administered DEHP
subcutaneously (0.99, 1.97, 4.93, or 9.86 gm/kg bw) on days 1, 5,
and 10; two groups of eight control mice each were given
subcutaneous injections of normal saline. On day 21 of the
experiment, each male was housed with one virgin female for seven
days. Female mice were sacrificed on day 13 of gestation: total
number of corpora lutea, implantations, early fetal deaths, and
viable fetuses were determined. The number of preimplantation
losses, an indirect indication of mutagenesis, was calculated from
the difference between the number of corpora lutea and
implantations in each animal. Increases in the incidences of
preimplantation losses and early fetal deaths were observed in
DEHP-treated groups. The authors concluded that calculated
mutagenic indices for control and DEHP-treated mice suggest a
dominant lethal mutation effect in treated mice.
Agarwal et al. (1986) investigated the effect of DEHP on
reproductive performance of male rats. Following dietary exposure
to DEHP and return to a normal diet for five days, twenty-four male
rats from each of five DEHP-dosage groups (0 to 20,000 mg/kg diet;
ppm) were housed individually with two sexually mature virgin
females (untreated) for five days. Mated females were housed
separately and allowed to litter naturally. Sixteen of the twenty-
four rats from each of the DEHP-dosage groups were maintained on a
normal diet for an additional 65 days, then mated for the
assessment of reproductive performance as described above. From
these experiments, the effects of DEHP on the incidence of
pregnancy, litter size, litter weight, and growth of pups up to 7
days of age were determined. The incidence of pregnancy, mean
litter weight on day 1, frequencies of stillbirths and neonatal
deaths and mean litter growth up to 7 days of age were unaffected
by DEHP treatment; however, mean litter size was significantly
reduced at 20,000 ppm DEHP. The authors suggested that these data
indicate a lack of reproductive dysfunction in F344 male rats at
DEHP doses below 20,000 ppm, a dose which also produced measurable
testicular degeneration and effects on sperm morphology.
Reproductive toxicity of DEHP in mice was assessed using the
National Toxicology Program (NTP) Fertility Assessment by
Continuous Breeding Protocol (Lamb et al., 1986; Melnick et
al., 1987). Male and female CD-1 mice were fed diets containing 0,
0.01, 0.1, or 3.0% DEHP during a 7-day pre-mating period and a
subsequent 98-day cohabitation period. For the continuous breeding
phase, males and females from the same dose group were randomly
paired and cohabitated for 98 days, with one breeding pair per
cage; the control group consisted of 40 pairs and each dose group
consisted of 20 pairs. After cohabitation, the breeding pairs were
separated and continued individually on treatment for 20 more days.
On each day of delivery, offspring were counted and weighed by sex,
then removed and killed. Continuous dietary exposure of CD-1 mice
to DEHP during the experiment resulted in complete suppression of
fertility in the 0.3% dose group and significant reduction of
fertility in the 0.1% group compared to the control group. In
addition, breeding pairs in the 0.1% treatment group had fewer male
and female live pups per litter and a lower proportion of pups born
alive per litter than did the breeding pairs in the control group.
There was no effect of DEHP on fertility in the 0.01% treatment
group.
In a crossover mating trial in which high-dose (0.3%) males
and females were randomly bred with control mice (animals were not
exposed to DEHP during this trial), the proportion of detected
matings did not differ among treatment groups; however, fertility
was significantly reduced for the 0.3% DEHP-treated males paired
with control males (0% fertility) versus the control × control
mating group (90% fertility). In addition, the proportion of pups
born alive was significantly lower in the 0.3% DEHP-treated males
paired with control females compared to the control × control
mating group. The authors concluded that, under the conditions of
these studies, DEHP was a reproductive toxicant in both male and
female CD-1 mice (Melnick et al., 1987).
In 1987, Melnick et al. (1987) reported the results of
studies by the National Toxicology Program (NTP); developmental
toxicity of DEHP was evaluated in Fischer 344 rats and in CD-1 mice
(Tyl et al., 1987). DEHP was administered to pregnant rats in the
diet at concentrations of 0, 0.5, 1.0, 1.5, or 2.0% on gestational
days 0 through 20. On gestational day 20, dams were killed and
evaluated for ovarian corpora lutea count, gravid uterine weight,
and status of uterine implantation sites. Live fetuses were
evaluated for viability, body weight, sex, gross morphological
abnormalities, and visceral or skeletal malformations. On
gestational day 20, mean absolute maternal weight gain (body weight
gain during gestation minus gravid uterine weight) was
significantly lower in the 1.0, 1.5. and 2.0% close groups compared
to the control group, and the mean gravid uterine weight was
decreased in the 2% dose group. There were no significant
differences in the number of implantation sites per litter in the
DEHP-treated pregnant rats compared to controls; however, the
percent resorptions per litter was significantly increased in the
2.0% dose group.
Treatment with DEHP did not affect the ratio of males to females,
but did cause a dose-related decrease in mean fetal body weight per
litter for both males and females. The number of fetuses malformed
per litter and the percent malformed fetuses were not significantly
different between DEHP-treated groups and the control groups. The
authors conclude that, at dose levels that caused maternal and
fetal toxicity (1.0 to 2.0%), DEHP did not produce increases in the
incidence of malformed rat fetuses.
In the NTP mouse developmental toxicity study, DEHP was
administered in the feed at concentrations of 0, 0.025, 0.05, 0.10,
or 0.15% on gestational days 0 through 17. Dose selection was based
on results from a preliminary toxicity study in pregnant CD-1 mice
in which 0.3% or higher DEHP in the diet caused maternal toxicity
(decrease in maternal weight gain) and 100% resorptions. Mean
absolute maternal weights were lower in the 0.10 and 0.15% dose
groups than in the control group. There were no dose-related
differences in the number of implantation sites per litter in the
DEHP-treated pregnant mice compared to controls; however, the
percent of resorptions per litter was significantly increased in
the 0.10 and 0.15% dose groups, the mean fetal body weight was
significantly reduced only in the 0.15% dose group compared to
controls. The percentage of fetuses with malformations and the
percentage of malformed fetuses per litter exhibited a significant
positive trend with 0.05-0.15% doses of DEHP. Gross defects
included eye and tail defects and exencephaly; visceral
malformations were predominantly aortic and pulmonary arch defects;
skeletal malformations included rib and thoracic central defects.
The authors concluded that DEHP was teratogenic to CD-1 mice when
administered in the feed at dose levels which produced maternal and
other fetal toxicity (0.10 and 0.15%) and at a dose level (0.05%)
which did not produce maternal or other fetal toxicity. At 0.025%
DEHP in the feed, there was no significant maternal or fetal
toxicity, including teratogenicity (Melnick et al., 1987).
Shiota & Mima (1985) reported on the teratogenicity of DEHP
and its principle metabolite, MEHP, in mice. Ten to 18-week old
female ICR mice were placed overnight with a male of proven
fertility. On days 7, 8, and 9 of pregnancy, DEHP (0, 250, 500,
1000, 2000 mg/kg bw) or MEHP (0, 50, 100, or 200 mg/kg bw)
dissolved in olive oil were administered by gavage or by
intraperitoneal injection. On day 18 of pregnancy, female mice were
killed and the number and position of implantations, resorptions,
and dead fetuses were recorded. Live fetuses were weighed, sexed,
and inspected for gross external abnormalities. Examination of live
fetuses for skeletal and internal soft tissue abnormalities was
omitted in this study because DEHP has been reported not to
increase these kinds of abnormalities in ICR mice (Shiota &
Nishimura, 1982). The authors reported that MEHP was more toxic to
female mice than DEHP. Among females that maintained their
pregnancy until term, there was no significant difference in the
average number of implants between control groups and groups given
DEHP or MEHP. The average weight of viable fetuses at term, however,
showed a dose-related decrease in groups receiving DEHP by gavage
(the decrease was significant at 100 and 2000 mg/kg). Oral
administration of DEHP increased the incidence of malformed viable
fetuses in a dose-related manner; anterior neural tube defects were
the malformations most commonly induced. The authors reported that
intraperitoneal administration of high doses of DEHP was
abortifacient, but that DEHP administered orally failed to exert
any notable teratogenic effects at doses below the abortifacient
doses. In addition, the authors reported that there was no
indication that either oral or intraperitoneal administration of
MEHP was teratogenic.
Tomita et al. (1986) reported that MEHP exerted embryo/
fetotoxic effects similar to those of DEHP at lower doses. Oral
administration of MEHP (1 ml/kg) to mice at 8 days of gestation
resulted in less than 32% live fetuses, all of which were
deformed. A single injection of MEHP (25 or 50 mg/kg), but not DEHP
(500 mg/kg), into pregnant mice induced a significantly higher
incidence of somatic mutations in the coat hair of the offspring.
The authors suggested that this data indicates that MEHP could be
responsible for the embryotoxic and fetotoxic effects observed with
DEHP. In contrast to this theory, Ritter et al. (1987)
hypothesize that DEHP exerts its teratogenic effects by in vivo
hydrolysis to 2-ethylhexanol, which in turn is metabolized to
2-ethylhexanoic acid, the proximate teratogen. When these three
agents were administered to Wistar rats on day 12 of gestation,
teratogenic responses indicated that they act through a common
mechanism and that, on an equimolar basis, DEHP was least potent,
2-ethylhexanol was intermediate in potency, and 2-ethylhexanoic
acid was the most potent teratogen.
Special studies on peroxisome proliferation and hepatocarcinogenicity
General hepatic effects of peroxisome proliferators
Dietary administration of compounds known to cause peroxisome
proliferation in the liver also cause liver hyperplasia and
hypertrophy. Enlargement of the liver begins shortly after the
peroxisome proliferator is first fed to the test animal. In
rodents, there is a gradual increase in liver size for 2 to 3
weeks; this final increased size is maintained for as long as the
peroxisome proliferator is administered. The liver returns to
normal weight within days after the peroxisome proliferator is
discontinued. Radioactive labeling and mitotic indices suggest that
the increase in liver weight is a genuine hyperplastic response
associated with the synthesis of new DNA. Both DEHP and its
principal hydrolysis product, MEHP, produce liver peroxisomal
proliferation and hypertrophy (National Toxicology Program, 1983;
Nair & Karup, 1986).
Smith-Oliver & Butterworth (1987) have reported the
correlation of the carcinogenic potential of DEHP with induced
hyperplasia rather than with genotoxic activity in the liver of
male mice. Genotoxicity was determined by the extent of DNA repair
(unscheduled DNA synthesis) and cell replication was determined by
the percentage of cells undergoing scheduled DNA synthesis.
Unscheduled and scheduled DNA synthesis were determined by
autoradiographic quantification of radiolabeled thymidine
incorporation in primary hepatocyte cultures treated directly or
isolated from adult (28-33 g) male B6C3F1 mice treated in vivo.
For the in vivo studies, DEHP was administered in the diet at a
concentration of 6000 ppm or given by gavage as a corn oil
solution (500 mg/kg); for the in vitro studies, cells were
incubated for 18 hours (1.5 hours after attachment) with DEHP
(0, 0.01, 0.1, or 1.0 µM) or MEHP (0, 0.1, 0.2, or 0.5 M) in
dimethyl sulfoxide. DNA repair was not detected in mouse
hepatocytes treated in vitro with DEHP or MEHP. DNA repair also
was not induced in hepatocytes isolated from mice treated with DEHP
for 12, 24, or 48 hours before sacrifice. However, a 15-fold
increase in the percentage of cells undergoing scheduled DNA
synthesis was observed in cells from male mice with 6000 ppm DEHP
in the diet for 7, 14, and 28 days did not stimulate the induction
of DNA repair in hepatocytes, however, the percentage of cells
undergoing scheduled DNA synthesis increased to over 9% at day 7
but returned to control values (0.3%) by day 14. The liver to body
weight ratio at day 28 was greater for DEHP-fed mice (8.3%) than
for control mice (4.8%).
The responses related to hyperplasia observed in DEHP-treated
B6C3F1 mice (Smith-Oliver & Butterworth, 1987) were greater than
those previously reported for F344 rats tested under similar
conditions (Butterworth et al., 1984). While a single dose of 500
mg/kg DEHP caused 0.6% of the cells to undergo scheduled DNA
synthesis in the rat, the same regimen caused 3.1% of the cells to
undergo scheduled DNA synthesis in the mouse. Female rats fed 6000
ppm DEHP in the diet for three weeks exhibited a liver to body
weight ratio that was 138% of controls, while the liver to body
weight ratio of male mice subjected to the same regimen was 173% of
controls.
Effects on liver peroxisomes, other subcellular organelles,
proteins, and enzymes
In addition to inducing liver hypertrophy and hyperplasia,
DEHP and other peroxisome proliferators have been reported to
affect liver peroxisomes, cytosol, mitochondria, microsomes, and
endoplasmic reticulum. Peroxisome proliferation is induced by a
wide variety of chemicals and conditions: hypolipidemic drugs,
phthalate esters, high-fat diet, and vitamin B deficiency, potency
of different proliferating agents varies over a 30-fold range.
Electron microscopy studies revealed a dose-related increase in rat
liver peroxisomes at DEHP dietary levels of 0.1% and above in males
and 0.6% and above in females (Chemical Manufacturers Association,
1987).
Mann et al. (1985) identified the major short-term hepatic
effects associated with administration of DEHP in the diet,
including midzonal and periportal accumulation of small droplets of
lipid, hepatomegaly accompanied by an initial burst of mitosis,
proliferation of hepatic peroxisomes and of the smooth endoplasmic
reticulum accompanied by induction of peroxisomal fatty acid
oxidation, damage to the peroxisomal enzymes as evidenced by
increased leakage of catalase to the cytosol, and centrolobular
loss of glycogen and decreases in glucose-6-phosphatase and low-
molecular weight reducing agents.
Tomaszewski et al. (1987) have asserted that acyl CoA
oxidase is a sensitive marker for early hepatic peroxisomal changes
caused by treatment of F344 rats with DEHP. Groups of five male
rats (250-300 g) were given 2.0 g/kg bw DEHP in corn oil daily for
1, 2, 3, 4, 7, or 14 days. Control rats received corresponding
amounts of corn oil (5 ml/kg). Acyl CoA oxidase activity was
increased 2.5-fold after 1 day and 8-fold after 14 days, enoyl CoA
hydratase activity increased 2-fold after 2 days and 6-fold after
14 days, there were no significant increases in hydroxyacyl CoA
dehydrogenase or catalase activities after 3 days of treatment with
DEHP. In a second experiment, rats were dosed with DEHP (0.06, 0.2,
0.6, 2.0, or 4.0 g/kg) dissolved in corn oil for 1, 3, or 7 days. At
the lowest dose, there was no significant increase in the relative
liver weight during the treatment period, at higher doses, increases
in relative liver weight were dose-related and significant. The
authors concluded that the apparent no-observable-effect level for
liver weight changes in response to DEHP treatment was 0.06 g/kg/day.
The effects of prolonged administration of DEHP on rat liver
were reported by Ganning et al. (1985). Male rats (180 g) were
fed DEHP in the diet (0, 0.02, 0.2, or 2.0%) for two years. Rats
were killed at intervals throughout the experiment, and changes in
KCN-insensitive palmitoyl-CoA dehydrogenase activity were monitored
as an indicator of general changes in peroxisomal beta-oxidation of
fatty acids. At the highest DEHP concentration (2.0% in the diet),
enzyme activity increased approximately 20-fold during 20 weeks;
slower but continuous elevations in activity were also obtained
with 0.2 and 0.02% DEHP, although activities after two years had
not reached the level achieved upon administration of 2.0% DEHP.
Among mitochondrial enzymes, those participating in fatty acid
transport (for example, carnitine-acetyl transferase) were markedly
induced during treatment with DEHP; other mitochondrial enzymes (NADH-
and NADPH-cytochrome c reductases, cytochrome b5, glucose-6-
phosphatase, and ATPase) were influenced only to a limited extent
or not at all. The cytochrome P-450 system was induced with the
highest dose of DEHP (2.0%), particularly during the early phase of
treatment. The authors emphasized that it is incorrect to speak of
threshold values for phthalate esters since intake of low amounts
for long periods of time can produce pronounced biological effects.
Preliminary studies have suggested that peroxisome
proliferation induced by MEHP, the principal metabolite of DEHP,
may be due to an initial biochemical lesion of fatty acid
metabolism resulting in increased intrahepatic lipid (Elcombe &
Mitchell, 1986). Evidence for this hypothesis comes from the
observation that increased omega- and beta-oxidation of fatty
acids, acyl CoA hydrolases, CoA, and carnitine are among the most
common observations following administration of peroxisome
proliferators to rodents and that similar changes in rodent liver
are affected by high-fat diets, where peroxisomal beta-oxidation
may be increased 8-fold (Reddy & Lalwani, 1983).
Watanabe et al. (1985) reported that DEHP induces marked
changes in the profile of some hepatic proteins. Reddy et al.
(1986a) reported that peroxisome proliferators ciprofibrate,
clofibrate, and DEHP selectively increased the rate of
transcription of the first two enzymes of the peroxisomal fatty
acid beta-oxidation genes (fatty acyl-CoA oxidase and enoyl-CoA
hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme) but
not of the catalase gene. Reddy et al., concluded that the
rapidity of the translational response (one hour after
administration of the test compounds) suggests that these agents
act directly on liver cells, and is reminiscent of receptor-
mediated responses. In partial support of this hypothesis, Lalwani
et al. (1983) have reported that the peroxisome proliferator
nafenopin binds to a cytosolic receptor in liver cells.
Current evidence indicates that maximal peroxisomal
proliferation is a tissue-specific phenomenon, restricted largely
to the hepatocyte but also reported to occur to a limited extend in
kidney and intestine. Reddy & Rao (1987) argue that the tissue-
specific nature of the biological response suggests that
interaction of these structurally dissimilar xenobiotics with a
receptor(s) might be the mechanism responsible for peroxisome
proliferation and the selective increase in the rate of
transcription of peroxisomal fatty acid beta-oxidation enzyme
system genes without significantly affecting the transcriptional
rate of peroxisomal marker enzyme catalase gene. They further
conclude that hepatic carcinogenicity of peroxisome proliferators
is not directly attributable to the chemical but to the adaptive
responses of the host (Reddy & Rao, 1987).
Nair & Kurup (1986) reported that dietary administration of
DEHP (2.0% w/w) for 30 days caused proliferation of hepatic
mitochondria in rats but not in Wistar mice. Inhibition of
respiratory activity on administration of DEHP was observed in
mitochondria isolated from the livers of rats, but not of mice.
DEHP administration increased the activity of alpha-glycero
phosphate dehydrogenase in hepatic mitochondria of the rat
(6-fold) and of the mouse (20%). Although the activity of carnitine
acetyltransferase was increased in liver mitochondria of both rats
and mice, the increase was greater in the rat (54-fold) than in
mice (36-fold). Nagi et al. (1986) reported that administration
of DEHP in the diet (2% v/w) for 8 days to male Sprague-Dawley rats
resulted in more than a three-fold increase in activity of acetyl
CoA-dependent hepatic mitochondrial fatty acid elongation, and that
this DEHP-sensitive activity was not due to peroxisomal contamination
of the mitochondrial fraction.
Ganning et al. (1985) reported that both decreased breakdown
and increased incorporation of amino acid precursors into rat liver
protein contribute to induction of mitochondria by DEHP
administration. The authors reported that DEHP appears to decrease
protein breakdown by interfering with membrane protein turnover,
and concluded that extensive induction of hepatic peroxisomes and
mitochondria during DEHP treatment is a complex process
characterized by both increased synthesis and decreased breakdown
of macromolecules.
Gollamudi et al. (1985) suggested that DEHP administration
may alter the composition of microsomal phospholipids in the liver
of male Sprague-Dawley rats: DEHP inhibited UDP-glucuronyl-
transferase activity of rat liver in vivo and in
vitro, but did not affect the activities of these cytosolic enzyme
N-acetyltransferase or microsomal P-450 in vitro.
Cook et al. (1986) demonstrated marked, yet differential,
stimulation of short-chain trans-2-enoyl CoA hydratase and
beta-ketoacyl CoA reductase activities in the liver peroxisomal,
microsomal, and cytosolic fractions from Sprague-Dawley rats
(200-250 g) treated with DEHP (2.0% v/w in the diet for 8 days).
Lake et al. (1987) studied the effects of prolonged
administration of clofibric acid and DEHP on lipid peroxidation in
the rat. Male Sprague-Dawley rats were fed diets containing either
0.05% clofibric acid (CA) or 2.0% DEHP for 2 years. Both compounds
produced liver enlargement accompanied by the formation of liver
nodules. Lipid peroxidation, as measured by whole homogenate
conjugated dienes, was increased to 620 and 640% of control levels
in livers from CA- and DEHP treated rats, respectively. The authors
concluded that prolonged peroxisome proliferation can result in
increased lipid peroxidation.
Goel et al. (1986) reported increased lipid peroxidation,
assessed by the measurement of conjugated dienes, in rats fed
ciprofibrate and Wy-14,642 - potent peroxisomal proliferators - for
6 months or longer. However, Prince et al. (1985) concluded that
lipid accumulation in the livers of male Wistar albino rats treated
with 25 mg/kg/day chlorpromazine did not induce peroxisome
proliferation by substrate overload.
Lake et al. (1987) observed that non-nodular regions of
DEHP-treated rat livers contained large deposits of mature
lipofucsin; Reddy & Lalwani have suggested that the formation of
lipofuscin is the result of sustained oxidative stress to the
hepatocytes (caused by increased generation of hydrogen peroxide),
which is manifested in enhanced lipid peroxidation (Reddy & Lalwani,
1983). In support of this hypothesis, peroxisome proliferators are
known to increase hepatocyte hydrogen peroxide levels (Goel et al.,
1986), peroxisomal fractions from treated animals exhibit an increased
capacity to generate highly reactive hydroxyl radicals (Elliot et
al., 1966), and antioxidants inhibit the carcinogenicity of the
potent peroxisome proliferator ciprofibrate (Rao et al., 1984).
Conway et al. (1987a, b) reported the results of experiments
designed to quantitate increases in conjugated dienes and
lipofuscin in the liver with long-term treatment of F344 rats with
peroxisome proliferators. Conjugated dienes and lipofuscin were
measured in livers from rats treated with DEHP and Wy-14643.
Conjugated dienes were increased about 45% in livers from rats
treated for 151 days with Wy-14,643; DEHP-treatment had no effect
on conjugated dienes. Autofluorescent lipofuscin was quantitated by
morphometry in sections of liver from control and treated rats.
Statistically significant increases in lipofuscin were observed
after 18 and 39 days of Wy-146432 and DEHP treatment, respectively.
At all time points up to 151 days following initiation of
treatment, the amount of lipofuscin in livers from Wy-14643-treated
animals was 5- to 10-fold more than in livers from DEHP-treated
animals. The authors suggested that these initial studies clearly
show a close association between the carcinogenicity of these two
peroxisome proliferating compounds and the accumulation of
lipofuscin: Wy-14643 in the diet caused nearly a 100% incidence of
hepatic cancer after 60 weeks (Rao et al., 1984), while DEHP in
the diet only caused a 10% incidence of hepatic cancer after two
years (National Toxicology Program, 1982).
Comparative effects of phthalates and phthalate metabolites
in various species
Significant increases in the number of peroxisomes and in the
activity of the hydrogen peroxide-generating peroxisomal fatty acid
beta-oxidation system occur in liver parenchymal cells of mice,
rats, and other species exposed to several structurally dissimilar
hypolipidemic drugs and certain phthalate ester plasticizers,
including DEHP (Reddy & Rao, 1987). Compared to other classes of
peroxisome proliferators, phthalate ester plasticizers, such as
DEHP, are relatively weak inducers of peroxisomal proliferation
(Moody & Reddy, 1978; Reddy et al., 1986b).
Mitchell et al., (1985a) reported the identification of
proximate peroxisome proliferators derived from DEHP. The authors
administered DEHP to rats and isolated the urinary metabolites;
major metabolites were ones resulting from initial omega or omega-1
oxidation of MEHP (metabolites were named according to the system
of Albro et al., 1973). These metabolites together with MEHP and
2-ethylhexanol, were added to primary rat hepatocyte cultures and
their effects on peroxisomal enzyme activity were observed.
Omega-oxidation products I and V and 2-ethylhexanol had little or
no effect on CN-insensitive palmitoyl-CoA oxidation (a peroxisomal
marker enzyme for beta-oxidation). Omega-1 oxidation products VI
and IX, as well as MEHP, produced large (7- to 11-fold) inductions
of peroxisomal enzyme activity. Similar results were observed for
the induction of cytochrome P-450-mediated lauric acid hydrolase
and increase in cellular coenzyme A content. The authors concluded
that the metabolites producing positive results in the
aforementioned assays represent proximate metabolites for DEHP-
induced peroxisome proliferation in rodent liver. Mitchell et al.
(1985b) also reported that oral administration of MEHP (150-250
mg/kg) to guinea pigs did not induce proliferation of hepatic
peroxisomes, and that addition of MEHP and other active metabolites
to primary guinea pig hepatocyte cultures failed to induce
peroxisomal beta-oxidation enzymes.
Mann et al. (1985) compared the short term effects of DEHP
to those of two straight-chain analogs in rats. It had previously
been reported that peroxisome proliferation was not induced in rats
administered free phthalic acid, nor in cultured hepatocytes
treated with n-hexanol (Gray et al., 1982a); the authors were
concerned with the question of whether straight-chain phthalates
differ in their hepatic effects from those of the branched chain
phthalates. Male Wistar albino rats (approximately 4 weeks old)
were allocated to 12 groups: Three control groups with 6 animals
each and 9 treatment groups with 4 animals each; three treatment
groups were assigned to DEHP, di(n-octyl)phthalate, and di(n-
hexyl)phthalate (administered in the diet at 20 g/kg; 20,000 ppm).
Groups of animals were killed 3, 20, and 21 days after the
beginning of treatment. The authors identified the major short-term
effects of administration of DEHP in the diet, many of which were
apparent 3 days after the initiation of treatment: (1) midzonal and
periportal accumulation of small droplets of lipid, (2)
hepatomegaly accompanied by an initial burst of mitosis, (3)
proliferation of hepatic peroxisomes and of the smooth endoplasmic
reticulum accompanied by induction of peroxisomal fatty acid
oxidation, (4) damage to the peroxisomal membranes as evidenced by
increased leakage of catalase to the cytosol, and (5) centrolobular
loss of glycogen and decreases in glucose-6-phosphatase
(endoplasmic reticulum-associated enzyme) and low-molecular weight
reducing agents. In contrast, diets containing the two straight-
chain phthalates induced (1) accumulation of large droplets of fat
around central veins leading, by 10 days, to mild centrolobular
necrosis, and (2) a very slight induction of catalase in the
peroxisomal fraction and a small, and late-appearing, increase in
liver weight; other changes induced by DEHP were not significantly
affected by the two straight-chain phthalates.
Benford et al. (1986) investigated differences in peroxisome
proliferation induced by MEHP and three longer branched chain
phthalates in hepatocytes isolated from adult male Wistar albino
rats and marmoset monkeys. Twenty-four hours after seeding, 0-0.6
µM MEHP, monoisonoyl phthalate, monoisodecyl phthalate, or di-isononyl
phthalate were added to the hepatocytes cultures; cultures were
analyzed for peroxisome proliferation, using increases in
peroxisomal palmitoyl-CoA oxidation as a marker. MEHP, monoisononyl
phthalate, and monoisodecyl phthalate produced marked dose-response
increases in peroxisomal palmitoyl-CoA oxidation in rat hepatocytes,
with MEHP producing the greatest response. In contrast, marmoset
peroxisomes showed only minimal changes with poor dose-response for
monoisononyl phthalate and monoisodecyl phthalate, and no change with
MEHP.
Lake et al. (1984) reported the results of comparative
studies of the hepatic effects of di- and mono-n-octyl
phthalates, DEHP, and the hypolipidemic drug clofibrate in male
Sprague-Dawley rats. DEHP (1000 mg/kg/day), its straight-chain
isomer di- n-octyl phthalate (DHOP; 1000 mg/kg/day), mono- n-
octyl phthalate (MNOP; 715 mg/kg/day), the straight-chain isomer of
MEHP, and clofibrate (500 mg/kg/day) were administered to 35 day
old rats by gavage for 14 days; control animals received 5 ml/kg bw
of the corn oil vehicle. All rats were starved overnight following
the last dose, then killed on day 15 of the experiment. Oral
administration of DNOP, MNOP, DEHP, and clofibrate to rats for 14
days produced significant increases in relative liver weight.
Marked peroxisome proliferation was observed in liver sections from
rats treated with DEHP and clofibrate, but not from rats treated
with DNOP and MNOP. DEHP and clofibrate, but not DNOP and MNOP,
produced marked increases in the hepatic activities of cyanide-
insensitive palmitoyl-CoA oxidation, a specific peroxisomal marker
enzyme, and carnitine acetyltransferase, located in mitochondrial,
peroxisomal, and microsomal fractions of liver cells. Only DEHP and
clofibrate treatment increased total (mitochondrial and
peroxisomal) and heat-labile (peroxisomal) enoyl-CoA hydratase
activities. While none of the compounds affected microsomal protein
content of the liver, both DEHP and clofibrate induced cytochrome
P-450 content; these two compounds, but not DNOP and MNOP, also
produced changes in the spectral properties of rat hepatic
microsomal hemoproteins.
Reddy et al. studied the induction of hepatic peroxisome
proliferation in nonrodent species, including primates (Reddy
et al., 1984). The hypolipidemic drug ciprofibrate, known to
induce peroxisome proliferation in rodent liver, was used in these
experiments. Male cats (500-700 g) were given ciprofibrate orally
in gelatin capsules (10-200 mg/kg bw) daily for up to 4 weeks;
pigeons were administered ciprofibrate by gavage (300 mg/kg bw) for
3 weeks; chickens received ciprofibrate (25-150 mg/kg bw) orally in
capsules for 4 weeks; adult male rhesus monkeys were allowed to eat
graded doses of ciprofibrate mixed with fruit jelly and bread
(50-200 mg/kg bw) for 7 weeks, and male cynomolgus monkeys were given
ciprofibrate in jelly and bread (400 mg/kg bw) for approximately 4
weeks. Treatment induced peroxisome proliferation in the livers of
all treated animals: cats at a dose greater than 40 mg/kg bw for 4
weeks; chickens at a dose greater than 25 mg/kg bw for 4 weeks;
pigeons at a dose of 300 mg/kg bw for 3 weeks; rhesus monkeys at
a dose of 50-200 mg/kg bw for 7 weeks; and cynomolgus monkeys at a
dose of 400 mg/kg bw for 4 weeks. In all five species studied, a
marked but variable increase in the activities of peroxisomal
catalase, carnitine acetyltransferase, heat-labile enoyl-CoA
hydratase, and the fatty acid beta-oxidation system was observed.
The authors concluded that peroxisome proliferation can be induced
in the livers of several non-rodent species, including primates,
and that it is a dose-dependent and not a species-specific phenomenon.
Hepatocyte transplantation systems are being used to evaluate
species differences in response to xenobiotic-induced peroxisome
proliferation (Reddy et al., 1984; Rao et al., 1986).
Enzymatically dissociated hepatocytes have been transplanted to the
interscapular or inguinal fat pads or to the anterior chamber of
the eye of a syngeneic host of an athymic nude mouse. The
transplanted hepatocytes responded to the peroxisome proliferative
effect of peroxisome proliferators administered in the diet of the
host. The degree of proliferation in transplanted liver was
comparable to the response observed in the parenchymal cells of the
homotropic liver. Hepatocytes from feline and canine livers, when
transplanted into athymic nude mice, appeared to respond to the
peroxisome proliferators administered to the nude mouse host, but
the magnitude of response was lower than that noted in the
hepatocytes of rats and mice.
In 1986, Elcombe & Mitchell reported that the exposure of
cultured rat hepatocytes to MEHP (0 to 0.5 mM) for 72 hours
resulted in marked induction of peroxisomal enzyme activity
(cyanide-insensitive palmitoyl CoA oxidase, an enzyme of the
peroxisomal beta-oxidation peroxisomes). Similar treatment of
cultured guinea pig, marmoset, or human hepatocytes revealed little
or no effect of MEHP. Identified proximate peroxisome proliferators
derived from MEHP (omega-1 oxidation products VI and IX, according
to the numbering system established by Albro et al. in 1973) were
also without effect in cultured guinea pig, marmoset, or human
hepatocytes.
In 1983, Gariot et al. (1983) reported that fenofibrate, a
triglyceride-lowering drug that also induces peroxisome
proliferation in rodent liver, failed to induce a similar response
in human liver. Ten patients (7 males, 3 females) with
hyperlipoproteinemia received fenofibrate at a daily dose of 300 mg
(6 subjects, 400 mg (2 subjects), and 600 mg (2 subjects) for from
16 days to 7.45 months (mean duration of treatment was 9.01
months). Thirteen patients in the control group (12 males, 1
female) affected by hyperlipoproteinemia were treated by diet only.
Liver biopsies were examined by light and electron microscopy; no
morphological differences were noted between the fenofibrate-
treated and control groups. The authors concluded that the
difference between this result and those consistently obtained in
rodents may be due to the relatively low doses given to human subjects
or to species dependent differences in liver response to peroxisome
proliferators. Similar examination of human liver biopsy material
from patients receiving peroxisome proliferating drugs gemfibrizol
and clofibrate also demonstrated marginal or no increases in
peroxisome numbers or volume densities (Blumcke et al., 1983; De
la Iglesia et al., 1982; Hanefeld et al., 1983).
In contrast top these results, Gunning et al. (1984)
reported slight peroxisome proliferation in human liver during
renal dialysis. However, the study did not present quantitative
data and patients were those with chronic renal dysfunction, which
has an unknown effect on liver peroxisomes.
Hepatic carcinogenesis: Initiation and promotion
It has been shown that seven hypolipidemic peroxisome
proliferators, including DEHP, induce hepatocellular carcinomas in
both rats and mice (Reddy et al., 1980; Reddy & Lalwani, 1983;
Rao et al., 1984). Liver rumours induced by peroxisome
proliferators are usually multiple, varying in size from small foci
of approximately 1 mm in diameter to large tumours of 20 to 40 mm
in diameters, and metastases in lungs are generally encountered
(Reddy & Rao, 1987). Rao et al. (1982) have shown that lesions
produced by peroxisome proliferators do not express gamma-glutamyl
transpeptidase activity; in addition, neoplastic liver lesions
induced in rats by peroxisome proliferators do not express the
placental form of gluthathione-s-transferase (Rao et al., 1986),
which has been shown to be a universal positive marker expressed in
liver lesions induced by genotoxic hepatocarcinogens (Sato et
al., 1984; Tatematsu et al., 1985). Reddy & Rao (1987) concluded
that expression of these two enzymes is not essential to the
initiation or promotion of liver carcinogenesis.
Reddy et al. (1986b) reviewed comparative morphometric and
biochemical data from rats treated with varying doses of
ciprofibrate, DEHP, and di(2-ethylhexyl)adipate (DEHA) and
concluded that the data indicate that the hepatocarcinogenic
potency of these agents is correlatable with their ability to
induce peroxisome proliferation, peroxisomal beta-oxidation, and
PPA-80, a peroxisome proliferation-associated 80,000 molecular
weight polypeptide. They also proposed a receptor mechanism for
induction of hepatic peroxisome proliferation by these and other
compounds.
Reddy et al. (1986) attempted to correlate the degree of
peroxisome proliferation in rat liver with the eventual
carcinogenic response. When rats were fed ciprofibrate (0.02%) or
DEHP (1.0%) in the diet for 30 days, peroxisome proliferation
induced by ciprofibrate was approximately double that induced by
DEHP. After treatment for one year, ciprofibrate in the diet (0.02%)
caused a 100% incidence of rats with hepatic carcinomas. The authors
suggested that the degree of peroxisome proliferation may be
predictive of the eventual carcinogenic response.
Researchers at CIIT further tested this hypothesis by
comparing the peroxisome proliferating effects and carcinogenicity
of Wy-14634 and DEHP in rats (Conway et al., 1987a). Wy-14643 in
the diet caused nearly a 100% incidence of male F344 rats with
hepatic neoplasia after 60 weeks (Rao et al., 1984); DEHP in the
diet only caused a 10% incidence of hepatic cancer after two years
(National Toxicology Program, 1982). When male F344 rats were fed
1.2% DEHP or 0.1% Wy-141643 in the diet for 1, 2, 4, 8, 39, 77, and
151 days, a near doubling of absolute liver weight was observed
within ten days. Similar increases in cell replication were
observed during the first days of treatment as measured by
autoradiographic quantification of nuclear labeling indices of
hepatocytes. Surprisingly, the induction of peroxisomal beta-
oxidation activity in liver homogenates by DEHP and Wy-14643 was
nearly identical: Both compounds increased peroxisomal beta-
oxidation activity by about 15-fold over controls after 18 days of
treatment and this level of induction was sustained until the end
of treatment (151 days). The authors concluded that, in direct
conflict with the suggestion of Reddy et al. (1986b) that the
degree of induction of peroxisome beta-oxidation enzymes can be
used to predict the relative carcinogenic effects of various
peroxisomal proliferators, effects of chronic DEHP and Wy-14643
treatment on the peroxisomal beta-oxidation system were not
predictive of the carcinogenicity of these two compounds.
Kluwe et al. (1985) and Kluwe (1986) studied the
relationship of the structure of phthalic acid esters and related
compounds to their carcinogenic potential in rats and mice. Groups
of 50 weanling F344 rats and B6C3F1 mice of each sex were exposed
to phthalic anhydride (PAn) [rat (male and female): 0, 7500, and
15,000 ppm; mouse (male): 0, 16,000, and 33,000 ppm; mouse
(female): 0, 12,000, and 24,000 ppm], DEHP [rat (male and female):
0, 6,000, and 12,000 ppm mouse; (male and female): 0, 3,000 and
6,000 ppm], DEHA [rat (male and female): 0, 12,000, and 25,000 ppm;
mouse (male and female): 0, 12,000, and 25,000 ppm], butyl benzyl
phthalate (BBP) [rat (female): 0, 6,000, and 12,000 ppm); mouse
(male and female): 0, 6,000, and 12,000 ppm], or 2-ethylhexyl
sulfate (EHS) [rats (male): 0, 10,000, and 20,000 ppm; mouse
(male): 0, 5,000, and 10,000 ppm; mouse (female): 0, 10,000, and
20,000 ppm] in the diet or diallyl phthalate (DAP) [rat (male and
female): 0, 50, and 100 mg/kg; mouse (male and female): 0, 150, and
300 mg/kg] or tris (2-ethylhexyl) phosphate (TEHP) [rat (male): 0,
2,000 and 4,000 mg/kg; rat (female): 0, 1,000 and 2,000 mg/kg;
mouse (male and female); 0, 500, and 1,000 mg/kg] with corn oil by
gavage (once per day, five days per week) for 104 weeks. Highest
doses given were estimated to be the maximum tolerable doses from
the results of 13-week studies. Except for DEHP (males: 10%
at high dose compared to 2% in controls and low-dose animals,
females: 16% at high dose compared to 4% in mid-dose and 0% in
control animals), none of the chemicals increased the incidence of
liver carcinomas in rats. However, the other three chemicals
with a 2-ethylhexyl moiety exhibited varying degrees of
hepatocarcinogenic activity in mice: DEHA increased the occurrence
of liver carcinoma in both male (24% for low and high doses
compared to 14% for controls) and female mice (28% for low dose and
24% for high dose compared to 2% for controls), although the
response was greater in females. TEHP caused a small but
significant increase in hepatocellular carcinomas in female mice
(0, 8, and 14% in control, low-, and high-dose groups,
respectively), while EHS equivocally increased hepatocellular
carcinomas in female mice (0, 2, and 7% in control, low-, and
high-dose groups, respectively). DEHP increased hepatocellular
carcinomas in mid(24%) and high-dose male mice (36%) compared with
male controls (18%), and in mid- (4%) and high-dose female mice
(34%) compared with female controls (0%). The 2-ethylhexyl compound
that evidenced the greatest hepatocarcinogenic response in mice
(DEHP) was also hepatocarcinogenic in rats; similarly, those
compounds with a relatively greater effect in female mice were also
active in male mice. The authors concluded that, although all of
the 2-ethylhexyl-containing compounds studied possessed some
hepatocarcinogenic activity, the results of these studies did not
reveal common neoplastic or non-neoplastic lesions suggestive of
structural correlates of toxic activity. Instead, sex and species
differences in 2-ethylhexyl-induced hepatocarcinogenesis in rodents
are probably quantitative rather than qualitative.
None of the carcinogenic peroxisome proliferators interact
with or damage DNA, suggesting that formation of a peroxisome
proliferator-DNA adduct is not an essential step in carcinogenesis
by this class of hepatocarcinogens (Von Daniken et al., 1984;
Goel et al., 1985; Gupta et al., 1985).
Gupta et al. (1985) attempted to identify peroxisome
proliferator-DNA adducts in rat liver cells under in vivo and
in vitro conditions. Clofibrate (250 mg/kg), ciprofibrate (50
mg/kg), Wy-14643 (50 mg/kg), and DEHP (2000 mg/kg) were
administered by gavage in 0.3 ml dimethyl sulfoxide to groups of
three F344 male rats (150 g) at 0, 24, and 48 hours after the
beginning of the experiment. Control rats received dimethyl
sulfoxide alone. Rats were killed 50 hours after the beginning of
the experiment. DNA isolated from the livers was analyzed for
possible carcinogen-DNA adducts by the 32P-postlabeling technique
which can detect one adduct in 100 billion nucleotides. Known DNA-
binding agents AAF and AAp served as positive controls. No adducts
were detected in the DNA isolated from livers of rats treated with
any of the peroxisome proliferators. For in vitro studies,
hepatocytes from male F344 rats were isolated, suspended in
chemically defined media containing 0.001 M clofibrate,
ciprofibrate, Wy-14643, or DEHP, and incubated for 4 hours.
Hepatocytes incubated in the presence of dimethyl sulfoxide served
as negative controls; hepatocytes incubated in the presence of AAF
(0.001 M) or N-OH-AAF (0.00001 M) served as positive controls. The
fact that adducts were not found in the DNA of hepatocytes exposed
in vitro to peroxisome proliferator-DNA adducts in hepatocytes by
this sensitive technique supports the contention that adduct
formation is not an essential step in carcinogenesis induced by
peroxisome proliferators.
Fahl et al. (1984) demonstrated DNA damage related to
increased generation of hydrogen peroxide by hypolipidemic drug-
induced liver peroxisomes. Peroxisomal-containing light
mitochondrial fractions were prepared from pooled livers of control
and hypolipidemic drug-treated rats (100 g male F344 rats were fed
0.1% Wy-14643 in the diet for 4 weeks). Drug treatment of animals
induced a large increase in the level of liver peroxisomal fatty
acid oxidation and an associated increase in the generation of
hydrogen peroxide. Hydrogen peroxide generation was the result of
an increase in the specific activity of peroxisomal hydrogen
peroxide-generating enzymes (6.3-fold) as well as an increase in
the number of peroxisomes per unit of liver volume (4.8-fold by
peroxisome recovery or 11-fold by monomorphic analysis of liver
sections). As a result, the overall peroxisome production of
hydrogen peroxide was increased 30- to 70-fold in the hypolipidemic
drug-treated rat livers, depending on the method used to assess the
numerical increase in peroxisome density. Peroxisomes isolated from
drug-treated rat liver, but not from control rat liver, induced
strand breaks and altered the electrophoretic mobility of
supercoiled SV40 DNA. Pure hydrogen peroxide also was able to
induce single strand nicks in a dose-dependent manner in SV40 DNA.
In incubations containing peroxisomes from drug-treated rats, a time-
and enzyme-dependent conversion to nicked SV40 DNA was observed
that paralleled the level of hydrogen peroxide production in the
peroxisome incubations. Catalase (400 units/ml) had only a marginal
quenching effect on the peroxisome-initiated DNA damage. The
authors suggested that these results are consistent with a
mechanism of hepatocarcinogenesis in which hepatocellular genetic
damage is introduced by the by-products of peroxisomal fatty acid
beta-oxidation, an oxidative pathway that is dramatically increased
in hypolipidemic drug-treated livers.
Rao et al. (1984) tested the hypothesis that antioxidants
could retard or inhibit neoplasia by scavenging active oxygen
(superoxide radicals, hydrogen peroxide, hydroxyl radicals, and
singlet oxygen). Groups of 25 male F344 rats were fed synthetic
antioxidants 2(3)-tert-butyl-14-hydroxyanisole (0.5% w/w) or
ethoxyquin (0.5% w/w) with or without the peroxisome proliferator
ciprofibrate (10 mg/kg bw) for 60 weeks. Rats fed ciprofibrate in
the diet or fed a diet with no added chemicals served as controls.
Ethoxyquin markedly inhibited the hepatic tumorigenic effect of
ciprofibrate, as evidenced by decreased incidence of rumours,
decreased number of tumours per liver, and reduced tumour size.
Administration of 2(3)-tert-butyl-14-hydroxyanisole also caused
a significant decrease in the incidence and number of hepatocellular
carcinomas larger than 5 mm. The authors postulated that the
inhibitory effect of these synthetic antioxidants on ciprofibrate-
induced hepatocarcinogenesis may be due to their hydrogen peroxide
and free radical-scavenging properties, since these antioxidants do
not prevent peroxisome proliferation and the induction of hydrogen
peroxide-generating peroxisomal enzymes in livers of rats fed
ciprofibrate.
Conway et al. (1987b) used oxidized glutathione (GSSG)
efflux into bile as an indicator of possible increases of hydrogen
peroxide concentrations in the extraperoxisomal compartment of
livers treated with nafenopin. Ackerboom et al. (1982) have
demonstrated that glutathione peroxidases form GSSG from reduced
glutathione during the detoxification of hydroperoxides and
hydrogen peroxide. Because glutathione peroxidases are located
outside the peroxisome in the cytoplasm and mitochondria, changes
in the efflux of GSSG into the bile can be used as a qualitative
measure of the diffusion of hydrogen peroxide out of peroxisomes.
Conway et al. infused various fatty acids into perfused livers
isolated from F344 male rats (150-300 g) treated with methyl-
cellulose (control; 1.0%) or nafenopin (80 mg/kg/day by
gavage for 7 days); GSSG efflux into the bile was measured. When
oleate (the predominant fatty acid found in rat blood) was infused,
a large efflux of GSSG into the bile of livers from nafenopin-
treated rats, but not control rats, was observed. Fatty acids with
varying substrate specificities for peroxisomal beta-oxidation were
tested: octanoate, laurate, and oleate excellent substrates - all
caused large increases in GSSG efflux from perfused livers of
nafenopin-treated, but not vehicle-control, rats; butyrate,
linoleate, and arachidonate with low activity for peroxisomal beta
oxidation - had no effect on GSSG efflux in perfused livers. Conway
et al (1987b) also demonstrated that bromooctanoate, an inhibitor
of fatty acid beta-oxidation, completely inhibited fatty acid-
induced increase in GSSG. The authors concluded that the data
suggest that some hydrogen peroxide produced by fatty acyl coenzyme
A oxidase during high rates of peroxisomal beta-oxidation in livers
from nafenopin-treated rats escapes detoxification by catalase and
diffuses into the cytoplasm to be metabolized by glutathione
peroxidase. The authors further suggested that these results
demonstrate a good correlation between substrate-specificity for
peroxisomal beta-oxidation and fatty acid-induced increases in GSSG
efflux.
Goel et al. (1986) have suggested that hydrogen peroxide
diffusing from the peroxisomes may be responsible for the
lipofuscin and conjugated dienes observed in liver after chronic
in vivo treatment with peroxisome proliferators.
Tomaszewski et al. (1986) reported increased peroxisomal
steady-state hydrogen peroxide levels in rats and mice treated with
peroxisome proliferators. Groups of 5 male F344 rats (250-350 g)
and female B6C3F1 mice (18-28 g) were dosed once per day for 14
days with DEHP (2 g/kg bw), DEHA (2 g/kg bw), or nafenopin (0.25
g/kg bw) dissolved in corn oil. Activities of enzymes responsible
for the production [peroxisomal palmitoyl CoA oxidase (PCO)] and
degradation [catalase (CAT) and glutathione peroxisome (GSHPx)] of
hydrogen peroxide were assayed in liver homogenates prepared from
treated and control animals. Activities of peroxisomal enzymes PCO
and CAT were enhanced 5- to 25-fold and 1.5- to 3-fold respectively
by treatment with peroxisome proliferators. The activity of
cytoplasmic GSHPx was reduced 40-60% in liver homogenates prepared
from treated animals compared to control animals. Treatment of rats
with peroxisome proliferators caused increases in steady-state
hydrogen peroxide in liver homogenates. The greatest increase
(approximately 13-fold) was produced by nafenopin; DEHA caused only
a 2-fold increase and DEHP produced a 7-fold increase relative to
control. In mouse liver homogenates, DEHP caused the greatest
increase (10-fold) in steady-state hydrogen peroxide relative to
control; nafenopin produced a 5-fold increase and DEHA caused a
2-fold increase. The authors also reported decreases in
concentrations of diene conjugates in the livers of treated
animals: In rats, the decrease corresponded with the increase in
steady-state hydrogen peroxide, but in mice this correspondence was
not observed. The authors concluded that the results of these
studies support the hypothesis that increased peroxisomal hydrogen
peroxide is involved in the hepatocarcinogenesis of peroxisome
proliferators.
Ward et al. (1983, 1986) reported on a number of studies on
the tumour initiating and promoting activities of DEHP in vitro and
in vivo. An initiation-promotion system for liver used male
B6C3F1 mice: To test for promotion, mice were injected with
diethylnitrosamine (DEN; 80 mg/kg bw) intraperitoneally once at 4
weeks; two weeks after injection, mice were placed on diets
containing 0, 3,000, 6,000, or 12,000 ppm DEHP or given water
containing phenobarbital (PB) at 500 ppm. At 2, 4, 6, 8, 10, or 18
months, groups of mice were killed; at selected times, hepatic DNA
synthesis and mitotic indices of hepatocytes were measured. To test
for initiating activity by DEHP, mice received one intragastric
dose (25 or 50 g/kg) at 4 weeks of age followed by PB (500 ppm in
drinking water) for 6 weeks; mice were killed at 6 and 18 months,
the authors reported that there was no evidence of liver tumour
initiation in mice by DEHP after 6 or 18 months of subsequent
exposure to the liver tumour promoter PB. However, both PB and DEHP
were effective tumour promoters. The focal hepatocellular
proliferative lesions (FHPL) in DEN-initiated mice that received
DEHP at 12,000 ppm were significantly larger in mean focus volume
at 6 months than those of mice in other groups; by 18 months, 25%
of the mice given 6000 ppm DEHP had hepatocellular carcinoma
metastatic to the lung.
In a separate experiment (Ward et al., 1984), DEHP was fed
in the diet at 3000 ppm or PB was given in the water at 500 ppm for
1, 7, 28, 84, or 168 days, beginning one week after DEN injection
at 4 weeks of age. All mice were killed at 168 days. Additional
groups of treated animals received DEHP or PB for 168 days and were
killed 84 days later to observe possible regression of hepatic
proliferative lesions. The authors concluded that DEHP was an
effective liver tumour promoter after 28, 84, and 168 days while PB
was effective only after 168 days of exposure. At 84 days after the
most prolonged period of exposure (168 days), however, FHPL in mice
given either PB or DEHP had not regressed but had increased in
size. The authors also reported that lung tumours were induced by
DEN in all groups of mice, but incidence of these tumours was not
affected by subsequent administration of either PB or DEHP.
Ward et al. (1986) also reported on the effectiveness of
DEHP as a tumour promoter in rat liver. Groups of 10 female F344
rats (5 weeks old) were injected intraperitoneally with N-
nitrosodiethylamine (282 mg/kg). Two weeks later, rats were placed
on diets containing 12,000 DEHP or on drinking water containing PB
at 500 ppm. After 14 weeks of exposure to the promoter, rats were
killed. By standard hematoxylin/eosin histology and histochemical
staining for gamma-glutamyl transpeptidase, DEHP failed to increase
the number or size of FHPL after 16 weeks, while PB was
significantly effective at the same doses used in mice.
Popp et al. (1984) also reported that DEHP lacked promoting
activity in rat liver. When DEHP was used in the promotion phase of
a rat (female CDF rats; 6-8 weeks old) hepatic initiation-promotion
system, no promoting activity could be demonstrated after 3 or 6
months of feeding DEHP at a dietary concentration of 1.2%. No liver
neoplasms or nodules were identified; in addition, DEHP did not
increase the number of foci or the mean volume of foci when foci
were identified by six different histologic stains.
Williams et al. (1987) reported that prolonged dietary
administration of DEHP (12,000 ppm for 24 weeks) to male F344 rats
(some of which had been initiated by N-2-fluorenylacetamide) did
not demonstrate initiating activity, significant sequential
syncarcinogenic activity, or a promoting effect on liver
carcinogenesis under conditions in which numerous agents with such
activities have been identified.
Garvey et al. (1987) reported that no initiating activity
was found when DEHP was administered in a single, oral dose (10
g/kg) or after 12 weeks of feeding by a promotion regimen. Animals
used were female F344 rats (150-180 g at the beginning of the
experiment). No liver tumours were found and there was no increase
in number of mean volume of loci when liver sections were examined
using multiple histologic markers.
DEHP was also tested for the ability to promote DMBA-induced
tumours in mouse skin (Ward et al., 1986). CD-1 mice initiated by
a single topical application of DMBA (50 µg) to the dorsal skin
received DEHP (98.1 µg in acetone, 0.2 ml total volume) or TPA (10
µg in 0.2 acetone) twice weekly for 40 weeks. To test for second-
stage promoting activity, female SENCAR mice were given DMBA once
(20 µg), and then TPA (2 µg twice a week for two weeks), followed
by DEHP (100 µg, twice weekly) or TPA, mezerein, or acetone weekly
for up to 26 weeks (Diwan et al., 1985). To test for complete
promoting activity by DEHP in SENCAR mice, DEHP was given twice
weekly after a single dose of DMBA (20 g) (Diwan et al., 1985).
The authors reported that DEHP did not promote the development of
skin tumours after DMBA initiation in CD-1 mice, nor was it an
initiator or complete skin carcinogen after 40 weeks. In female
SENCAR mice, however, DEHP was a weak second-stage promotor (6.4
papillomas per mouse compared to 0 for control mice initiated by
DMBA) and a weaker complete promoter of skin carcinogenesis (0.9
papillomas per mouse). Mezerein was a stronger second-stage
promoter (23 papillomas per mouse) and TPA was a stronger complete
promoter (26.4 papillomas per mouse).
Ward et al. (1986) and Diwan et al. (1985) also reported
on the results of in vitro studies designed to test promoting
activity of DEHP. Cultures of three epidermis-derived cell lines of
the JB6 mouse (C141, C121, and R219) were suspended in medium
containing DEHP (1.3-51.2 × 10-3 M in acetone), MEHP (1-5 × 10-3
M in DMSO), or 2-ethylhexanol (EH; 4-7 × 10-4 M in DMSO), Suspensions
were layered over agar; colonies in agar were counted 14 days later.
These cell lines have previously been shown to be promoted to
anchorage independence by tumour promoting phorbol esters and also by
mezerein, nezoyl peroxide, and epidermal growth factor. DEHP showed
activity for promotion of transformation in all three of these cell
lines: C141 gave the most pronounced response to DEHP, with nearly
32% of cells giving rise to colonies after treatment with 2.7 × 10-7
M DEHP. MEHP was also shown to be an effective promoter; EH,
however, failed to promote transformation in this system.
Numoto et al. (1984) described the effects of clofibrate and
nafenopin on the rat liver cell membrane enzymes gamma-glutamyl
transpeptidase and alkaline phosphatase during the early stages of
hepatocarcinogenesis. Male F344 rats (8 weeks of age) were fed
diets containing 0.02% N-2-fluorenylacetamide (FAA) for 8 weeks to
induce hepatocellular altered foci, and were than given no
chemical, equimolar amounts (0.03 mmol/kg diet) of clofibrate or
nafenopin, or 0.07% of the liver promoter phenobarbital (PB) in the
diet for 24 weeks. PB had a marked enhancing effect on FAA-induced
loci, while clofibrate produced only slight enhancement and
nafenopin produced none. Nafenopin suppressed histochemical gamma-
glutamyl transpeptidase activity in the abnormal hepatocytes of the
foci as well as in periportal hepatocytes. In homogenates of lives
from rats fed nafenopin and, to a lesser extent, clofibrate,
activity of this enzyme was reduced, whereas PB enhanced its
activity. The authors suggested that these results reveal
significant cell membrane effects of nafenopin and clofibrate and
suggest that their involvement in hepatocarcinogenesis is more
complex than a promoting action.
DeAngelo & Garret (1983) and DeAngelo et al. (1984) reported
the inhibition of phenobarbital- and dietary choline deficiency
promoted preneoplastic lesions on rat liver by DEHP. Gamma-glutamyl
transpeptidase positive (GGT+) preneoplastic foci were initiated in
the liver of Sprague-Dawley male rats given a single dose of
diethylnitrosamine (DEN) following partial hepatectomy. Promotion
of loci occurred by one of three methods: (1) a choline-deficient
diet (CD), (2) a choline-supplemented diet containing 0.6%
phenobarbital (CS+PB), and (3) a Cd diet containing 0.06%
phenobarbital (CD+PB). In rats receiving DEN, each promoting
regimen effectively increased the number of GGT+ loci above levels
in control rats fed only the CS diet. Inclusion of 2.0% DEHP in
each of the dietary promotion treatments, however, effectively
inhibited the appearance of the loci. However, DeAngelo et al.
later reported that DEHP was unable to inhibit the promoting effect
of the CD diet at a concentration of 0.1% (DeAngelo et al.,
1985b), and that di-n-octyl phthalate, a relatively ineffective
peroxisome inducing straight chain isomer of DEHP, enhances the
development of initiated GGT+ loci in rat liver (DeAngelo et al.,
1986).
DeAngelo et al. (1985a) postulated that DEHP may inhibit
emergence of GGT+ foci in initiated rats by blocking the response
of initiated cells to stimulation by epidermal growth factor: DEHP
in the diet of rats (2.0%) blocked the ability of epidermal growth
factor to enhance the phosphorylation of its receptor protein in
isolated liver plasma membranes.
Perera & Shinozuka (1984) and Perera et al. (1987)
investigated the relationship between a CD diet and peroxisome
proliferators on liver rumour promotion. A CD diet, Which is an
efficient liver tumour promoter, induced peroxidative damage of
liver cell membrane lipids. By modifying components of the CD diet,
Perera et al. were able to demonstrate that the efficacy of the
promotion is correlated with the extent of lipid peroxidation. Both
an antioxidant and hypolipidemic peroxisome proliferators BR931 and
DEHP suppressed CD diet-induced lipid peroxidation and promoting
effects in rats initiated with diethylnitrosamine.
However, Hagiwara et al. (1986) reported that butylated
hydroxianisole (BHA), an antioxidant, had no effect on liver tumour
promotion by DEHP. Four week-old male B6C3F1 mice were given a
single intraperitoneal injection of N-nitrosaodiethylamine (DEN; 80
mg/kg) followed one week later by oral exposure to DEHP (6000 ppm
in the diet) and/or butylated hydroxianisole (7500 ppm in the diet) for
29 weeks. DEHP and BHA (alone and together) increased the incidence of
DEN-initiated focal hepatocellular proliferative lesions, including
both microscopic hyperplastic foci and hepatocellular adenomas.
COMMENTS AND EVALUATION
DEHP-induced testicular atrophy in rats is an age-dependent
response, younger rats being more susceptible. Although zinc-
deficient rats showed enhanced susceptibility to the gonadotoxic
effects of DEHP, testicular atrophy was not prevented by the
co-administration of zinc with DEHP. The co-administration of
testosterone with DEHP to adult male rats appears to prevent
testicular injury otherwise induced by DEHP. Mono-2-ethylhexyl
phthalate (MEHP), a metabolite of DEHP, is likely to be the active
metabolite of DEHP affecting rat testes both in vivo and in
vitro. There is a partial reversibility of DEHP-induced testicular
atrophy in rats following cessation of exposure to DEHP.
The hepatocarcinogenicity in rats and mice attributed to DEHP
and other phthalate esters is preceded by hepatocellular peroxisome
proliferation. Suggestions as to the probable mechanism of
peroxisome proliferation have been advanced but remain unproved.
It was noted that the lowest DEHP level in food contact
materials is a function of its effectiveness in plasticizing
(softening) plastic materials. Technologically optimum DEHP levels
range from 20 to 50% of the weight of the plastic material.
Migration levels of DEHP into food are influenced by its level in
the packaging material and factors such as the composition of food
and time and temperature of processing and storage of packaged
food. As a consequence of its extreme solubility in lipid
materials, the use of DEHP-containing materials in some countries
is limited to foods of high water content (non-fatty foods).
After reviewing all the information available, the Committee
reiterated its recommendation that human exposure to DEHP is food
be reduced to the lowest level attainable. The Committee considered
that this might be achieved by using alternative toxicologically
acceptable plasticizers or alternatives to plastic materials
containing DEHP.
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