DNA damage in tissues of rat treated with potassium canrenoate

Potassium canrenoate (PC), a competitive aldosterone antagonist previously found to increase tumor incidence in rats and to produce genotoxic effects in in vitro systems, was examined in rats to acquire information on its genotoxic activity in vivo. Intragastric administration of 1/2 LD50 produced, as revealed by the Comet assay, a modest but statistically significant increase in the frequency of DNA lesions in liver but not in thyroid and bone marrow of male rats, and in thyroid and bone marrow but not in liver of female rats. In contrast with the frankly positive responses observed in primary cultures of rat hepatocytes (Martelli et al., Mutagenesis 14 (1999) 463-472) any evidence of DNA repair and micronuclei formation was absent in liver of rats treated with 1/2 LD50, and initiation of enzyme-altered liver preneoplastic lesions did not occur in the liver of rats given 100 mg/kg PC once a week for 6 successive weeks. A high and dose-dependent frequency of DNA lesions was found to occur in testes and ovaries of rats given single doses ranging from 1/8 to 1/2 LD50.     

Saccharomyces cerevisiae intervention for relieving flutamide-induced hepatotoxicity in male rats

The aim of this work was to investigate the protective role of baker's yeast Saccharomyces cerevisiae against the hepatotoxic effect of the drug flutamide that is widely used for treatment of metastatic prostate adenocarcinoma. Administration of flutamide to adult male rats in a dose of 100 mg/kg b.w. daily for 15 days resulted in serious hepatic injury. Highly significant increase in each of serum ALT, ALP, bilirubin, bile acids and cholesterol level, relative to the control group, was observed. Also, a highly significant increase in the serum glutathione-S-transferase isoforms: alpha-GST and pi-GST and each of TNF-alpha and NO levels was recorded. Moreover, highly significant decrease in hepatic glutathione peroxidase and superoxide dismutase activities was observed. In addition, the authors noticed a significant increase in serum testosterone levels with concomitant highly significant increase in serum acid phosphatase activity. Prophylactic treatment of male rats with baker's yeast in a dose of 4.8 mg/kg b.w. daily for 15 days, followed by a combination of flutamide (100 mg/kg b.w.) and yeast (4.8 mg/kg b.w.) daily for other 15 days resulted in marked improvement in rat's liver function, whereas the serum testosterone and acid phosphatase levels retained values parallel to those recorded for the flutamide-treated rats. Histological examination of liver tissues showed that flutamide caused hydropic degeneration, necrotic areas and marked increase in Kupffer cells. The central vein is congested with blood and signs of apoptosis appeared in the hepatocytes in the form of fragmentation of the nuclei and blebbing of the cytoplasm. On the other hand, in the rats treated with both yeast and flutamide, the hepatic cords were more regularly arranged, signs of degeneration or apoptosis were less pronounced and some hepatocytes appeared binucleated. The authors postulate that each one of the powerful antioxidative components in S. cerevisiae effectively participated in attenuation of the oxidative stress caused by flutamide metabolites, and in promoting regeneration of new hepatocytes and meanwhile could restore liver function beyond normal status.     

The effect of caffeine on diethylnitrosamine-initiated hepatic altered foci in a mid-term induction system.

The modification potentials of caffeine on the development of preneoplastic hepatic enzyme altered foci were examined in an in vivo mid-term assay system. The number and area of glutathione S-transferase placental form-positive (GST-P+) hepatic foci was significantly reduced in rats given caffeine (0.1% or 0.2% in drinking water) followed by diethylnitrosamine (DEN) (200 mg/kg BW, IP) and DEN followed by caffeine as compared with the controls given carcinogen alone. Unscheduled DNA synthesis (USD) decreased approximately 70% in the hepatocytes treated with caffeine (200 mg/ml of medium). These results suggested that the antiinitiative effect of caffeine might be caused by the inhibition of the intracellular carcinogen accumulation and the antipromotive effect of caffeine might be associated with suppression of DNA repair.     

The effect of different diets or mineral oil on liver pathology and polybrominated biphenyl concentration in tissues

Groups of 10 adult (3.5 months) male Sherman strain rats were given 0 or a single dose of 500 mg polybrominated biphenyls (PBB) in corn oil/kg by stomach tube. Three weeks later, groups of 10 rats each that had received 500 mg/kg and groups of 10 rats each that had been given corn oil were started on a purified 20% fiber or 4% fiber diet. Ten rats each that had received 500 mg/kg or corn oil were continued on Purina Chow. An additional group of 10 rats given 500 mg/kg was also continued on Purina Chow but was given 2 ml mineral oil/kg three times a week. All rats were given the different diets or the mineral oil for 3 months. Chemical analysis of blood, liver, and adipose tissue by gas chromatography for PBBs showed no statistically significant difference in the PBB concentrations in blood and adipose tissue among the different groups. The PBB concentrations in the liver of rats fed Purina Chow were significantly lower than in the other groups if they were calculated on a wet weight basis. When PBB concentrations in liver were calculated on a lipid basis, the differences were not statistically significant. Total liver lipids showed statistically significant increases in rats given 500 mg/kg followed by 4 or 20% purified fiber diet or by Purina Chow and 2 ml of mineral oil/kg every other day. Microscopic examination of the livers showed essentially normal parenchyma in all rats that had not received PBBs. The rats given a single oral dose of 500 mg/kg which was followed by Purina Chow or Purina Chow and mineral oil showed some centrolobular vacuolated hepatocytes and enlarged hepatocytes around the central veins. The vacuolation was more pronounced in rats receiving Purina Chow and mineral oil also. Rats given 500 mg/kg followed by 4 or 20% purified fiber diet showed severe steatosis of the liver with megalo-hepatocytes, areas of cell necrosis and interstitial fibrosis. The interstitial fibrosis was more pronounced in rats given the 20% purified fiber diet. These results indicate a pronounced additive effect on PBB liver toxicity in rats by the 4 or 20% purified fiber diets which may be deficient in some nutrients or may prevent absorption of important nutrients.     

DNA damage induced by 4,4'-methylenedianiline in primary cultures of hepatocytes and thyreocytes from rats and humans

4,4'-Methylenedianiline (MDA), an aromatic amine used in various industrial processes and previously found to induce tumor development in liver and thyroid of mice and rats, was evaluated for its DNA-damaging activity in primary cultures of hepatocytes and thyreocytes from rat and human donors. After exposure for 4 and 20 h to MDA concentrations ranging from 10 to 180 microM, a statistically significant increase in the frequency of DNA lesions was revealed by the Comet assay in primary hepatocytes and thyreocytes from donors of both species, the response being dose dependent up to 56-100 microM MDA. DNA fragmentation was more marked after 4 than after 20 h exposure in all four cell types. DNA was damaged to a lesser extent in human hepatocytes and thyreocytes than in corresponding rat cells and in both species in hepatocytes than in thyreocytes. In both rat and human hepatocytes a 20-h exposure to the same MDA concentrations elicited a modest amount of DNA repair synthesis, as evaluated by autoradiography. Evidence of a partial reduction of DNA damage, and therefore of only partial DNA repair, was observed in rat hepatocytes and in rat and human thyreocytes incubated for 16 h in MDA-free medium after a 4-h MDA treatment. A 4-h exposure to 56, 100, and 180 microM MDA did not induce DNA lesions in primary cultures of cells from three rat organs, kidney, urinary bladder mucosa, and brain, which are resistant to MDA carcinogenic activity. Under the same experimental conditions any evidence of DNA damage was absent in primary kidney and urinary bladder cells from human donors. Taken as a whole the results of this work indicate that MDA is specifically activated to DNA-damaging reactive species by hepatocytes and thyreocytes in both rats and humans and thus suggest that liver and thyroid might be the targets of the carcinogenic activity of MDA also in humans.     

DNA damage induced by 3,3'-dimethoxybenzidine in liver and urinary bladder cells of rats and humans.

3,3'-Dimethoxybenzidine (DMB), a congener of benzidine used in the dye industry and previously found to be carcinogenic in rats, was evaluated for its genotoxic activity in primary cultures of rat and human hepatocytes and of cells from human urinary bladder mucosa, as well as in liver and bladder mucosa of intact rats. A similar modest dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic DMB concentrations ranging from 56 to 180 microM. Replicating rat hepatocytes displayed a modest increase in the frequency of micronucleated cells after a 48-h exposure to 100 and 180 microM concentrations. In primary cultures of human urinary bladder mucosa cells exposed for 20 h to 100 and 180 microM DMB, the Comet assay revealed a clear-cut increase of DNA fragmentation. In rats given one-half LD50 of DMB as a single oral dose, the GSH level was reduced in both the liver and urinary bladder mucosa, whereas DNA fragmentation was detected only in the bladder mucosa. Taken as a whole, these results suggest that DMB should be considered a potentially genotoxic chemical in both rats and humans; the selective effect on the rat urinary bladder might be the consequence of pharmacokinetic behavior.     

Chronic toxicity and oncogenicity studies of ingested 1, 3-dichloropropene in rats and mice

Fischer 344 rats and B6C3F1 mice were administered 1, 3-dichloropropene (1,3-D) via their diets for up to 2 years, at dose levels of 0, 2.5, 12.5, or 25 mg 1,3-D/kg body wt/day for rats and 0, 2.5, 25, or 50 mg 1,3-D/kg body wt/day for mice. The test material was stabilized in the feed by microencapsulation in a starch/sucrose matrix (80/20%). Rats given 12.5 or 25 mg/kg/day, and mice given 25 or 50 mg/kg/day, had decreased body weights and body weight gains. There were no effects on survival or clinical pathology parameters for rats or mice. Histopathologic effects attributed to treatment in rats consisted of basal cell hyperplasia of the nonglandular mucosa of the stomach in males and females given 12.5 or 25 mg/kg/day for 12 and 24 months and an increased number of hepatocellular adenomas in males given 12.5 or 25 mg/kg/day and females given 25 mg/kg/day for 24 months. The increase in hepatocellular adenomas was statistically identified by pairwise comparison only in males given 25 mg/kg/day. An increased incidence of eosinophilic foci of altered cells in the liver was also noted in all treated groups of rats at 24 months. The latter observation, however, was considered of equivocal toxicological significance because of the common spontaneous occurrence of liver foci in aged Fischer 344 rats. The only histologic change attributed to treatment in mice was decreased size of hepatocytes in males given 50 mg/kg/day for 12 months. The decreased size of hepatocytes was consistent with decreased cytoplasmic glycogen content and corresponded to decreased liver weights. This effect was not present at 24 months. There was no oncogenic response observed in mice. The low-dose level of 2.5 mg/kg/day was interpreted as the no-observed-adverse-effect level (NOAEL) for systemic chronic toxicity of 1,3-D in the Fischer 344 rat. The no-observed-effect level (NOEL) for chronic systemic toxicity was 2.5 mg/kg/day in the B6C3F1 mouse.     

Pathophysiological role of poly(ADP-ribose) polymerase (PARP) activation during acetaminophen-induced liver cell necrosis in mice.

DNA fragmentation in hepatocytes occurs early after acetaminophen (AAP) overdose in mice. DNA strandbreaks can induce excessive activation of poly(ADP-ribose) polymerases (PARP), which may lead to oncotic necrosis. Based on controversial findings with chemical PARP inhibitors, the role of PARP-1 activation in AAP hepatotoxicity remains unclear. To investigate PARP-1 activation and evaluate a pathophysiological role of PARP-1, we used both PARP inhibitors (3-aminobenzamide; 5-aminoisoquinolinone) and PARP gene knockout mice (PARP-/-). Treatment of C3Heb/FeJ mice with 300 mg/kg AAP resulted in DNA fragmentation and alanine aminotransferase (ALT) release as early as 3 h, with further increase of these parameters up to 12 h. Few nuclei of hepatocytes stained positive for poly-ADP-ribosylated nuclear proteins (PAR) as indicator for PARP-1 activation at 4.5 h. However, the number of PAR-positive cells and staining intensity increased substantially at 6 and 12 h. Pretreatment with 500 mg/kg 3-aminobenzamide before AAP attenuated hepatic glutathione depletion and completely eliminated DNA fragmentation and liver injury. Delayed treatment several hours after AAP was still partially protective. On the other hand, liver injury was not attenuated in PARP-/- mice compared to wild-type animals. Similarly, the specific PARP-1 inhibitor 5-aminoisoquinolinone (5 mg/kg) was not protective. However, 3-aminobenzamide attenuated liver injury in WT and PARP-/- mice. In summary, PARP-1 activation is a consequence of DNA fragmentation after AAP overdose. However, PARP-1 activation is not a relevant event for AAP-induced oncotic necrosis. The protection of 3-aminobenzamide against AAP-induced liver injury was due to reduced metabolic activation and potentially its antioxidant effect but independent of PARP-1 inhibition.     

Trichloroethylene effects on the formation of enzyme-altered foci in rat liver

The initiating and promoting effects of trichloroethylene in rat liver were investigated using the enzyme-altered foci bioassay. The incidence of gamma-glutamyl transpeptidase (GGT)-positive foci was used as an early histochemical marker of putative preneoplastic hepatocytes. A single PO dose of trichloroethylene (490 mg/kg) was administered in corn oil to rats which had been partially hepatectomized 24 h previously. Three days following gavage with the chlorinated hydrocarbon the rats were promoted with an 8-week regimen of 500 ppm phenobarbital in drinking water. This protocol is known to induce enzyme-altered foci in the livers of animals which have received an initiating dose of a genotoxic carcinogen. Trichloroethylene was not found to induce GGT-positive foci under these conditions. Additionally, groups of rats were partially hepatectomized, initiated with N-nitrosodiethylamine (30 mg/kg; PO) and administered five times weekly doses of 200 mg trichloroethylene per rat in order to investigate the promoting activity of the chlorinated hydrocarbon in rat liver. No significant promoter effects were observed with trichloroethylene, although the results in this case were somewhat equivocal. The findings of these investigations are taken as partially supportive of an epigenetic, cytotoxic mechanism of tumorigenic action of trichloroethylene.     

Assessment of unscheduled and replicative DNA synthesis in hepatocytes treated in vivo and in vitro with unleaded gasoline or 2,2,4-trimethylpentane

In a recent chronic inhalation exposure study, unleaded gasoline (UG) produced kidney tumors in male rats and liver tumors in female mice, but did not increase the incidence of liver tumors in male mice or rats of either sex. To examine the possible basis for this pattern of hepatocarcinogenesis, unscheduled DNA synthesis (UDS) as an indicator of genotoxic activity and replicative DNA synthesis (RDS) as an indicator of cell proliferation were measured in rat and mouse hepatocytes following in vivo and in vitro exposures to UG and 2,2,4-trimethylpentane (TMP), a nephrotoxic component of UG. Primary hepatocyte cultures, prepared from cells isolated from Fischer-344 rats, B6C3F1 mice, or human surgical material, were incubated with [3H]thymidine and the test agent. UDS was measured by quantitative autoradiography as net nuclear grains (NG). By similar methods, UDS and RDS (S-phase cells) were measured in hepatocytes isolated from rats and mice treated by gavage with TMP (500 mg/kg) or UG (100 to 5,000 mg/kg). A dose-related increase in UDS activity was observed in rat hepatocytes treated in vitro with 0.05 to 0.10% (v/v) UG. These doses were, however, toxic in both mouse and human hepatocyte cultures. Weak UDS activity was observed in hepatocytes isolated from male and female mice treated 12 hr previously with UG. No UDS was induced in rat hepatocytes treated in vivo or in vitro with TMP. Twenty- and fourfold increases in the percentage of cells in S-phase were observed 24 hr after treatment with TMP in male and female mice, respectively, as compared to a fivefold increase in male rats. UG increased the percentage of S-phase cells in male mice by ninefold but failed to induce RDS in females. Thus, there appears to be genotoxic compounds in UG that can be detected in cultured hepatocytes and in the livers of exposed mice. The lack of UDS activity in rat liver was consistent with the reported lack of liver tumors in chronically exposed rats. However, neither the UDS nor the RDS responses in mice exposed by gavage correlated to the sex-specific pattern of liver tumors observed in the 2-year bioassay.     

Involvement of bile salt export pump in flutamide-induced cholestatic hepatitis

The non-steroidal antiandrogen flutamide is widely used for treatment of prostatic cancer, but causes side effects, including cholestatic hepatitis and fulminant hepatitis. We investigated the pathogenesis of flutamide-induced cholestatic hepatitis, focusing on the bile salt export pump (BSEP; ABCB11), which exports bile salts to the bile. We examined the inhibitory effects of flutamide and its active metabolite, hydroxyflutamide, on the transport of taurocholic acid (TCA) by membrane vesicles derived from hBSEP-expressing Sf9 cells. Flutamide inhibited the transport of TCA by hBSEP (IC50 value, about 50 microM), while hydroxyflutamide had no effect at up to 100 microM. When flutamide was administered to rats as a single oral dose of 100 mg/kg, the biliary excretion rate of bolus-injected [3H]TCA was decreased and the liver tissue concentration of flutamide exceeded 50 microM. Repeated doses of flutamide for 5 d (10 mg/kg/d) also decreased the biliary excretion rate of bolus-injected [3H]TCA. In this case, the liver tissue concentration of flutamide was below 0.1 microM. In both cases, no change in the mRNA level of rat Bsep was detected by RT-PCR. These results suggest that flutamide itself, but not its major metabolite, may cause cholestasis by inhibiting BSEP-mediated bile salt excretion.     

Evaluation of cultured, precision-cut rat liver slices as a model to study drug-induced liver apoptosis

The precision-cut liver slice culture model has been used widely to investigate drug metabolism and drug-induced necrosis. However, apoptosis, a key mediator of liver toxicity remains to be studied in this model. We evaluated apoptosis induced by thioacetamide (TAA) in rat liver slices, and in livers taken from TAA-treated rats as a control. Rat liver slices were treated with 50, 75 and 100 mM of TAA for 15 h. Histopathological examination of the liver slices revealed specific centrilobular localization of apoptotic hepatocytes at 75 mM but randomly distributed at 100 mM. Apoptosis in centrilobular hepatocytes was confirmed by appearance of cleavage products of caspase-3 and DNA fragmentation studied by TUNEL method. A concentration-dependent release of cytochrome c was observed in the slices, suggesting a role for mitochondria in the apoptosis triggered by TAA. The in vitro results were compared to the data obtained in male Sprague-Dawley rats given a single ip injection of 40 mg/kg TAA and sacrificed 1, 2, 3 and 6 h after dosing. Histopathological analyses showed specific centrilobular localization of apoptosis after 6 h treatment. Caspase-3 activation, DNA fragmentation and cytochrome c release were also observed in the liver of rats treated with TAA. Overall these data indicated that precision-cut liver slices provide a valuable in vitro system to study drug-induced liver apoptosis.     

Evaluation of in vivo genotoxic potential of fenofibrate in rats subjected to two-week repeated oral administration

Fenofibrate (FF), a peroxisome proliferator-activated receptor-alpha agonist, has been used as one of the hypolipidemic drugs in man and induces oxidative stress and promotes hepatocarcinogenesis in the liver of rodents. This chemical belongs to a class of non-genotoxic carcinogens, but DNA damage secondary to oxidative stress resulting from reactive oxygen species (ROS) generation is suspected in rodents given this chemical. To examine whether FF has genotoxic potential, partially hepatectomized F344 male rats were treated orally with 0, 1,000 or 2,000 mg/kg of FF for 2 weeks, followed by diet containing 0.15% 2 acetyl aminofluorene (2 AAF) for enhancement the tumor-promoting effect for 10 days and a single oral dose of carbon tetrachloride (CCl4) as the first experiment (liver initiation assay). As the second experiment, the in vivo liver comet assay was performed in hepatectomized rats, and the expression of some DNA repair genes was examined. In the liver initiation assay, the number and area of glutathione S-transferase placental form (GST-P)-positive single cells and foci did not increase in the FF treated groups. In the comet assay, positive results were obtained after 3 h of the last treatment of FF, and the expression of some DNA repair genes such as Apex1, Ogg1 and Mlh1 were upregulated in rats given the high dose of FF at 3 h after the treatment but not in 24 h after the treatment. The results of the present study suggest that FF causes some DNA damage in livers of rats, but is not a strong genotoxic substance leading to a DNA mutation since such DNA damage was repaired by the increased activity of some DNA repair genes.     

Pyridine does not induce unscheduled DNA synthesis (UDS) in hepatocytes of male B6C3F1 mice treated in vivo

Pyridine was evaluated in an in vivo/in vitro mouse DNA repair assay. Unscheduled DNA synthesis (UDS) was used as an indicator of DNA damage to hepatocytes from male B6C3F1 mice. Test animals were exposed by oral gavage to pyridine or to the vehicle or positive control articles, and hepatocytes were collected and labeled by incubation in media supplemented with [3H]thymidine. Following labeling, the cultures were processed for autoradiographic analysis. Doses were selected based on a pilot study in which 0, 250, 500, 750, 1000 or 2000 mg kg(-1) pyridine in water was administered by gavage. Mice in the 1000 and 2000 mg kg(-1) dose groups were comatose following dosing and died within 24 h of dose administration. Pyridine dose levels for the UDS determination were set at 175, 350 and 700 mg kg(-1). Pyridine solutions in water were administered to mice 2 or 16 h prior to the scheduled sacrifice. The vehicle control group received water 16 h before sacrifice and the positive control group received 10 mg kg(-1) dimethylnitrosamine (DMN) 2 h before sacrifice. Pyridine did not significantly increase the UDS response in hepatocytes isolated from the treated animals, as measured by the incorporation of [3H]thymidine, using standard criteria for a negative response: less than zero mean net grains in repair (NG) and <20% of cells in repair (% IR; cells in repair have at least 5 NG). The vehicle control group and the low, mid- and high pyridine dose groups yielded less than -8.3 NG and < or =1% IR. The positive control group yielded a positive UDS response, with 10.8 NG and 62% IR. These results indicate that pyridine is non-genotoxic in B6C3F1 mouse liver using the UDS endpoint.     

Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide

Gestational and lactational exposure to di(n-butyl) phthalate (DBP) at >/=250 mg/kg/day disrupts male rat reproductive development and function. Although this indicates an antiandrogenic mechanism, DBP and its biologically active metabolite do not interact with the androgen receptor (AR) in vitro. In the present study, we compared the effects of DBP and the antiandrogen flutamide using a shorter exposure during the prenatal period of male sexual differentiation in rats. Pregnant CD rats received DBP at 0, 100, 250, or 500 mg/kg/day po (n = 10) or flutamide at 100 mg/kg/day po (n = 5) from Gestation Days 12 to 21. In F1 males, DBP (500 mg/kg/day) and flutamide caused hypospadias; cryptorchidism; agenesis of the prostate, epididymis, and vas deferens; degeneration of the seminiferous epithelium; and interstitial cell hyperplasia of the testis. Flutamide and DBP (250 and 500 mg/kg/day) also produced retained thoracic nipples and decreased anogenital distance. Interstitial cell adenoma occurred at 500 mg DBP/kg/day in two males. The only effect seen at 100 mg DBP/kg/day was delayed preputial separation. In contrast to flutamide, DBP caused a low incidence of prostate agenesis and hypospadias with no vaginal pouch. The low incidence of DBP-induced intraabdominal testes contrasted with the high incidence of inguinal testes seen with flutamide. Thus prenatal male sexual differentiation is a sensitive period for the reproductive toxicity of DBP. A no observed adverse effect level was not established and the lowest observed (adverse) effect level was 100 mg/kg/day. Flutamide and DBP disrupted the androgen signaling necessary for male sexual differentiation but with a different pattern of antiandrogenic effects. DBP is an example of an environmental antiandrogen that disrupts androgen-regulated male sexual differentiation without interacting directly with the AR, as does flutamide.     

Male Reproductive Tract Malformations in Rats Following Gestational and Lactational Exposure to Di(n-butyl) Phthalate: An Antiandrogenic Mechanism?

Di(n-butyl) phthalate (DBP), a widely used plasticizer suspectedof having estrogenic properties, was investigated for its effectson the prenatal and early neonatal development of the reproductivetract. Pregnant CD rats (n = 10) were given DBP at 0, 250, 500,or 750 mg/kg/day (p.o.) throughout pregnancy and lactation untiltheir offspring were at postnatal day 20. Maternal body weightsthroughout the dosing period were comparable in all groups.At 750 mg/kg/day, the number of live pups per litter at birthwas decreased and maternal effects on pregnancy and postimplantationloss are likely to have occurred. Anogenital distance was decreasedat birth in the male offspring at 500 and 750 mg/kg/day. Theepididymis was absent or underdeveloped in 9, 50, and 71% ofadult offspring (100 days old) at 250, 500, and 750 mg/kg/day,respectively, and was associated with testicular atrophy andwidespread germ cell loss. Hypospadias occurred in 3, 21, and43% of males and ectopic or absent testes in 3,6, and 29% ofmales at 250, 500, and 750 mg/kg/day, respectively. Absenceof prostate gland and seminal vesicles as well as small testesand seminal vesicles were noted at 500 and 750 mg/kg/day. Vaginalopening and estrous cyclicity, both estrogen-dependent events,were not affected in the female offspring, although low incidencesof reproductive tract malformations were observed at 500 and750 mg/kg/day. In the male offspring, DBP produced the samespectrum of effects elicited by the antiandrogen flutamide.Thus, DBP specifically impaired the androgen-dependent developmentof the male reproductive tract, suggesting that DBP is not estrogenicbut antiandrogenic in the rat at these high dose levels. Forhuman risk assessment, determining if this toxicity is metabolite-mediatedwill be critical, since marked species differences in metabolismexist.     

Ongoing hepatocellular regeneration and resiliency toward galactosamine hepatotoxicity

In previous studies, we reported that the age-dependent hepatotoxicity of galactosamine (GalN) was evident in hepatocytes maintained in primary cultures. Cellular proliferation and tissue repair are not manifested in response to injury in this in vitro system. Neonatal (5-day) rats have ongoing hepatocellular proliferation in contrast to adult (5-month) rats, and should be therefore resilient to GalN toxicity. Liver injury was assessed by serum transaminases (ALT, AST),³H-thymidine (³H-T) incorporation into nuclear DNA, and content of hepatocellular nuclear DNA. While the dose of 400 mg/kg did not cause any significant liver injury in the neonates, it did produce significant liver injury in adult rats. At a dose of 800 mg/kg, GalN produced significant injury in the neonates. Because 400 mg/kg causes clearly demonstrable liver injury in the adult and no injury in the neonates, this dose was used for further studies. In addition to the above measures of injury, uracil nucleotides (UTP, UDP, and UMP), glycogen, histopathology, and autoradiographic examination of liver sections were used to assess the liver injury in neonatal and adult rats. In a time-course study, all of the above were measured at 0, 12, 24, 36, 48 and 72 h after GalN administration. Serum enzyme elevations as well as the appearance of necrotic and swollen hepatocytes were maximal at 24 h in the adults rats. In contrast to these observations in the adult rats, none of these measurements indicated significant liver injury in the neonates.³H-T incorporation into nuclear DNA was much higher in the neonatal liver in comparison to the adults reflecting the difference in regeneration. Hepatocellular nuclear DNA was also higher in the neonate and was significantly decreased due to GalN treatment. In the adult rats, the quiescent normal level of³H-T incorporation and nuclear DNA content were further decreased at 12 h, increased at 48 h and returned to normal low, quiescent levels at 72 h. In the neonates mitotic activity of hepatocytes was higher than in the adult rats. In the adult rats, mitotic activity was increased at 48 h after GalN administration and returned to normal at 72 h. In the neonates GalN did not alter the mitotic activity significantly. These findings demonstrate that in the presence of hepatocellular regeneration, galactosamine toxicity is minimal while in the absence of it, clear toxicity is manifested. In conclusion, while perturbation in uracil nucleotides and related biochemical events may explain the infliction of liver injury by GalN in an age-dependent fashion, the extent of tissue repair impacts decisively on the final outcome of injury.A preliminary report of these findings was presented at the 30th Annual Meeting of the Society of Toxicology at Dallas, TX. The Toxicologist 11: 169 (1991)Recipient of the 1988 Burroughs Wellcome Toxicology Scholar Award.     

A 52-week repeated dose toxicity study of ultraviolet absorber 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)benzotriazole in rats

A 52-week repeated dose toxicity study of an ultraviolet absorber, 2-(2'-hydroxy-3',5' -di-tert-butylphenyl)benzotriazole (HDBB), was conducted according to OECD TG 452 under GLP. CD(SD)IGS rats were given HDBB by gavage at 0, 0.1, 0.5, or 2.5 mg/kg/day in males and 0, 0.5, 2.5, or 12.5 mg/kg/day in females. No substance-related deaths or clinical signs of toxicity were observed in any group; however, a lowered body weight was found from day 36 to the end of the 52-week administration period at 2.5 mg/kg in males. At the completion of the dosing period, a decrease in red blood cells at 0.5 mg/kg and higher, and in hematocrit at 2.5 mg/kg, was detected in males. Blood biochemical changes, including increases in the levels of alkaline phosphatase and glucose and the A/G ratio, were also found at 0.5 mg/kg and higher in males and at 12.5 mg/kg in females. At necropsy, absolute and relative liver weight was increased at 0.5 mg/kg and higher in males and at 12.5 mg/kg in females. Histopathological changes were observed in the liver; centrilobular hypertrophy of hepatocytes at 0.5 mg/kg and higher in males, and at 12.5 mg/kg in females, and altered hepatocellular foci at 0.5 mg/kg and higher, and cystic degeneration and lipofuscin deposition in hepatocytes at 2.5 mg/kg in males. Based on these findings, the no observed adverse effect level was concluded to be 0.1 mg/kg/day in male rats and 2.5 mg/kg/day in female rats.