The effects of subchronic acrylamide exposure on gene expression, neurochemistry, hormones, and histopathology in the hypothalamus-pituitary-thyroid axis of male Fischer 344 rats.

Acrylamide (AA) is an important industrial chemical that is neurotoxic in rodents and humans and carcinogenic in rodents. The observation of cancer in endocrine-responsive tissues in Fischer 344 rats has prompted hypotheses of hormonal dysregulation, as opposed to DNA damage, as the mechanism for tumor induction by AA. The current investigation examines possible evidence for disruption of the hypothalamic-pituitary-thyroid axis from 14 days of repeated exposure of male Fischer 344 rats to doses of AA that range from one that is carcinogenic after lifetime exposure (2.5 mg/kg/d), an intermediate dose (10 mg/kg/d), and a high dose (50 mg/kg/d) that is neurotoxic for this exposure time. The endpoints selected include: serum levels of thyroid and pituitary hormones; target tissue expression of genes involved in hormone synthesis, release, and receptors; neurotransmitters in the CNS that affect hormone homeostasis; and histopathological evaluation of target tissues. These studies showed virtually no evidence for systematic alteration of the hypothalamic-pituitary-thyroid axis and do not support hormone dysregulation as a plausible mechanism for AA-induced thyroid cancer in the Fischer 344 rat. Specifically, there were no significant changes in: 1) mRNA levels in hypothalamus or pituitary for TRH, TSH, thyroid hormone receptor alpha and beta, as well 10 other hormones or releasing factors; 2) mRNA levels in thyroid for thyroglobulin, thyroid peroxidase, sodium iodide symporter, or type I deiodinases; 3) serum TSH or T3 levels (T4 was decreased at high dose only); 4) dopaminergic tone in the hypothalamus and pituitary or importantly 5) increased cell proliferation (Mki67 mRNA and Ki-67 protein levels were not increased) in thyroid or pituitary. These negative findings are consistent with a genotoxic mechanism of AA carcinogenicity based on metabolism to glycidamide and DNA adduct formation. Clarification of this mechanistic dichotomy may be useful in human cancer risk assessments for AA.

Levels of retinyl palmitate and retinol in the skin of SKH-1 mice topically treated with retinyl palmitate and concomitant exposure to simulated solar light for thirteen weeks.

Retinyl esters account for more than 70% of the endogenous vitamin A found in human skin, and retinyl palmitate is one of the retinyl esters in this pool. Human skin is also exposed to retinyl palmitate exogenously through the topical application of cosmetic and skin care products that contain retinyl palmitate. To date, there is limited information on the penetration and distribution of retinyl palmitate and vitamin A within in the skin. In this study, the accumulation of retinyl palmitate and generation of retinol in the skin of male and female SKH-1 mice that received repeated topical applications of creams containing 0.0%, 0.1%, 0.5%, 1.0%, 5.0%, 10%, or 13% of retinyl palmitate 5 days a week for a period of 13 weeks were studied. Because products containing retinyl palmitate are frequently applied to sun-exposed skin, and because it is well established that exposure to sunlight and UV light can alter cutaneous levels of retinoids, mice in this study were additionally exposed 5 days a week to simulated solar light. The results showed that retinyl palmitate diffused into the skin and was partially hydrolyzed to retinol. The levels of retinyl palmitate in the skin of mice that were administered retinyl palmitate cream were higher than control values, and levels of both retinyl palmitate and retinol increased with the application of higher concentrations of retinyl palmitate in the cream. Our results indicate that topically applied retinyl palmitate may alter the normal physiological levels of retinyl palmitate and retinol in the skin of SKH-1 mice and may have a significant impact on vitamin A homeostasis in the skin.

Effect of dietary genistein on cell replication indices in C57BL6 mice.

The phytoestrogen and isoflavone, genistein, inhibited the activity of the DNA synthesis-related enzyme, topoisomerase-II (topo-II), altered cell-cycle traverse and produced cell death in cell culture models. In order to examine the potential effects of genistein on cell replication and cell death in an animal model, 8-week-old C57BL6 mice were fed either a control diet or one containing one of five doses (100-2000 ppm) of genistein for 28 days. At the end of the feeding period, both male and female mice were sacrificed and the serum isoflavone and aglycone levels determined by liquid chromatography with electrospray tandem mass spectrometry (LC-ES/MS/MS). Immunohistochemistry was utilized to measure the cell replication and cell death rates in the small intestine. Total isoflavone concentration increased from below the limits of detection (0.001 microM) in control animals to 0.28 microM in male and 0.54 microM in female mice fed the 2000 ppm diet. A decrease in the percentage of cells in G(0) and an increase in the percentage of cells in S-phase, consistent with topo-II-induced S-phase arrest, was found in the duodenum and jejunum of the small intestine. Thus, genistein appears to accumulate to a sufficient level to affect topo-II activity in the small intestine.

Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley) rats.

Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. A dose range-finding study was conducted as a prelude to a multigeneration bioassay to assess potential toxicities associated with genistein consumption. Genistein was administered in a soy- and alfalfa-free diet at 0, 5, 25, 100, 250, 625, or 1250 ppm to pregnant dams starting on Gestation day 7 and continuing throughout pregnancy. Dietary exposure of the dams continued through lactation, and pups were maintained on the same dosed feed as their mother after weaning until sacrifice at Postnatal day 50. Body weight and feed consumption of the treated dams prior to parturition showed a decreasing trend with a significant reduction at the highest dose. Litter birth weight was depressed in the 1250 ppm dose group, and pups of both sexes in that dose group had significantly decreased body weights relative to controls at the time of sacrifice. The most pronounced organ weight effects in the pups were decreased ventral prostate weight in males at the 1250 ppm dose and a trend toward higher pituitary gland to body weight ratios in both sexes. Histopathologic examination of female pups revealed ductal/alveolar hyperplasia of the mammary glands at 250 to 1250 ppm. Ductal/alveolar hyperplasia and hypertrophy also occurred in males, with significant effects seen at 25 ppm and above. Abnormal cellular maturation in the vagina was observed at 625 and 1250 ppm, and abnormal ovarian antral follicles were observed at 1250 ppm. In males, aberrant or delayed spermatogenesis in the seminiferous tubules relative to controls was observed at 1250 ppm. There was a deficit of sperm in the epididymis at 625 and 1250 ppm relative to controls, although testicular spermatid head counts and epididymal spermatozoa counts did not show significant differences from controls at these doses. Both sexes showed an increase in the incidence and/or severity of renal tubal mineralization at doses of 250 ppm and above. Dietary genistein thus produced effects in multiple estrogen-sensitive tissues in males and females that are generally consistent with its estrogenic activity. These effects occurred within exposure ranges achievable in humans.

Acetaminophen-induced hepatotoxicity in mice lacking inducible nitric oxide synthase activity.

We recently reported that nitrotyrosine and acetaminophen (APAP)-cysteine protein adducts colocalize in the hepatic centrilobular cells following a toxic dose of APAP to mice. Whereas APAP-adducts are formed by reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine, nitrotyrosine residues are formed by reaction of tyrosine with peroxynitrite. Peroxynitrite is formed from nitric oxide (NO) and superoxide. This manuscript examines APAP (300 mg/kg) hepatotoxicity in mice lacking inducible nitric oxide synthase activity (NOS2 null or knockout mice; C57BL/6-Nos2(tm1Lau)) and in the wildtype mice. In a time course the ALT levels in the exposed NOS2 null mice were approximately 50% of the wildtype mice; however, histological examination of liver sections indicated similar levels of centrilobular hepatic necrosis in both wild-type and NOS2 null mice. Serum nitrate plus nitrite levels (NO synthesis) were identical in saline-treated NOS2 null and wild-type mice (53 +/- 2 microM). APAP increased NO synthesis in wild-type mice only. The increases paralleled the increases in ALT levels with peak levels of serum nitrate plus nitrite at 6 h (168 +/- 27 microM). In wild-type mice hepatic tyrosine nitration was greatly increased relative to saline treated controls. Tyrosine nitration increased in NOS2 null mice also, but the increase was much less. APAP increased hepatic malonaldehyde levels (lipid peroxidation) in NOS2 null mice only. The results suggest the presence of multiple pathways to APAP-mediated hepatic necrosis, one via nitrotyrosine, as in the wild-type mice, and another that is not dependent upon inducible nitric oxide synthase activity, but which may involve increased superoxide.

Basal cell proliferation in female SKH-1 mice treated with alpha- and beta-hydroxy acids.

Alpha- and beta-hydroxy acids are compounds that have been used extensively in cosmetic and dermatological formulations. Clinical and qualitative effects of alpha- and beta-hydroxy acids have been well characterized, but little is known about their mechanism of action or acute and chronic biochemical effects. In the present study, we examined the acute proliferative effects of glycolic and salicylic acids on cell proliferation in the epidermis of SKH-1 female mice, using BrdU incorporation as a marker of epidermal proliferation. In preliminary experiments, we observed an increase in the rate of proliferation after 3 days of treatment with 10% glycolic acid-containing cream and this was sustained throughout a 6.5-week (treatment 5 days/week) time course compared with untreated control animals. After each treatment with cream containing glycolic acid there was a wave of proliferation that was maximal 12 to 16 h (significant at p < 0.05) after treatment, followed by a subsequent increase in epidermal thickness at 18 to 20 h (significant at p < 0.05). The effects of the concentration and pH level of glycolic acid- and salicylic acid-containing creams on the rate of proliferation and increases in skin thickness in SKH-1 epidermis were also investigated. We observed a dose-dependent increase in epidermal proliferation of animals treated with either glycolic or salicylic acid. A similar time-dependent response was observed in the epidermal thickness in animals treated with salicylic acid, but not with glycolic acid. Differences in pH (3.5 or 4.0) had no significant effect on either epidermal proliferation or skin thickness. The data that we present here should be useful in characterizing not only the beneficial but also the adverse effects that occur following acute or chronic usage of alpha-hydroxy acids.

Diallyl sulfide inhibition of CYP2E1 does not rescue diabetic rats from thioacetamide-induced mortality.

Previously we have shown that hepatotoxicity of thioacetamide (TA) was increased in streptozotocin (STZ)-induced diabetic (DB) rats due to combined effects of enhanced bioactivation-based liver injury of TA and compromised liver tissue repair response. We have also shown that TA is primarily bioactivated by hepatic CYP2E1. The present study was done to further investigate the importance of liver tissue repair in determining the final outcome of hepatotoxicity. STZ-induced DB rats were pretreated with a CYP2E1 inhibitor, diallyl sulfide (DAS), to decrease the bioactivation-based liver injury of TA. The treatments were as follows: DB/DAS/TA, DB/corn oil/TA, and DB/DAS/saline. Nondiabetic (non-DB) rats received the same treatments as controls. A dose of TA (300 mg/kg ip), which was nonlethal in non-DB rats, caused 92 and 90% mortality in DB/DAS/TA and DB/corn oil/TA groups, respectively. At various times (0--60 h) after treatment, liver injury was assessed by plasma alanine aminotransferase and histopathology. Cell proliferation was evaluated by [(3)H]thymidine incorporation and immunohistochemical staining of proliferating cell nuclear antigen (PCNA). In the DB/DAS/TA rats, DAS pretreatment markedly reduced the CYP2E1-dependent liver injury of TA compared to that in DB/corn oil/TA rats. However, subsequent hepatic DNA synthesis in both DB groups was inhibited approximately 50%. PCNA analysis showed a corresponding decrease in cell-cycle progression. This compromised tissue repair response in DB rats was insufficient to compensate for cell loss, resulting in progression of liver injury and culminating in high mortality in both DB groups. Furthermore, non-DB rats were pretreated with a CYP2E1 inducer, isoniazid, to increase the bioactivation-based TA liver injury equal to the liver injury observed in DB/DAS/TA rats. Despite equal injury up to 36 h following TA treatment, the tissue repair response in the non-DB rats was highly stimulated to compensate for liver injury and led to 70% survival in this group. These studies underscore the importance of adequate and timely tissue repair in compensating for liver injury and protecting from lethality.

Fumonisin b1 carcinogenicity in a two-year feeding study using F344 rats and B6C3F1 mice.

Fumonisin B1 (FB1) is a mycotoxin isolated from Fusarium fungi that contaminate crops worldwide. A previous study demonstrated that FB1 promoted preneoplastic foci in initiated rats and induced hepatocellular carcinomas in BD IX rats at 50 parts per million (ppm), but fundamental dose-response data were not available to assist in setting regulatory guidelines for this mycotoxin. To provide this information, female and male F344/N/Nctr BR rats and B6C3F1 Nctr BR mice were fed for two years a powdered NIH-31 diet containing the following concentrations of FB1: female rats, 0, 5, 15, 50, and 100 ppm; male rats, 0, 5, 15, 50, and 150 ppm; female mice, 0, 5, 15, 50, and 80 ppm; male mice, 0, 5, 15, 80, and 150 ppm. FB1 was not tumorigenic in female F344 rats with doses as high as 100 ppm. Including FB1 in the diets of male rats induced renal tubule adenomas and carcinomas in 0/48, 0/40, 9/48, and 15/48 rats at 0, 5, 15, 50, and 150 ppm, respectively. Including up to 150 ppm FB1 in the diet of male mice did not affect tumor incidence. Hepatocellular adenomas and carcinomas were induced by FB1 in the female mice, occurring in 5/47, 3/48, 1/48, 19/47, and 39/45 female mice that consumed diets containing 0, 5, 15, 50, and 80 ppm FB1, respectively. This study demonstrates that FB1 is a rodent carcinogen that induces renal tubule tumors in male F344 rats and hepatic tumors in female B6C3F1 mice.

Compensatory regeneration as a mechanism for renal tubule carcinogenesis of fumonisin B1 in the F344/N/Nctr BR rat.

Fumonisin B1(FB1) is a fungal metabolite of Fusarium verticillioides (= F. moniliforme), a fungus that grows on many crops worldwide. Previous studies demonstrated that male BD IX rats consuming diets containing 50 ppm fumonisin B1 developed hepatocellular carcinomas. In our recent studies, diets containing FB1 at 50 ppm or higher concentrations induced renal tubule carcinomas in male F344/N/Nctr BR rats and hepatocellular carcinomas in female B6C3F1/Nctr BR mice. The carcinogenicity of FB1 in rats and mice is not due to DNA damage, as several laboratories have demonstrated that FB1 is not a genotoxin. FB1 induces apoptosis in cells in vitro. Including FB1 in the diets of rats results in increased hepatocellular and renal tubule epithelial cell apoptosis. In studies with F344/N/Nctr BR rats consuming diets containing up to 484 ppm FB1 for 28 days, female rats demonstrated more sensitivity than male rats in the induction of hepatocellular apoptosis and mitosis. Conversely, induction of renal tubule apoptosis and regeneration were more pronounced in male than in female rats. Induction of renal tubule apoptosis and hyperplasia correlated with the incidence of renal tubule carcinomas that developed in the 2-year feeding study with FB1 in the F344/N/Nctr BR rats. The data are consistent with the hypothesis that the induction of renal tubule carcinomas in male rats could be partly due to the continuous compensatory regeneration of renal tubule epithelial cells in response to the induction of apoptosis by fumonisin B1.

Cellular origins of ultraviolet radiation-induced corneal tumours in the grey, short-tailed South American opossum (Monodelphis domestica).

Corneal tumours were induced in almost 100% of grey, short-tailed South American opossums (Monodelphis domestica) exposed three times weekly to ultraviolet radiation (UVR) for periods of a year or more. Five tumours, representing the morphological spectrum of UVR-induced corneal tumours (two fibrosarcomas, one malignant fibrous histiocytoma, one putative haemangiosarcoma, and one squamous cell carcinoma overlying a sarcoma), were assayed immunohistochemically for reactivity with antibodies against the intermediate filaments vimentin, smooth muscle actin (alpha isoform), muscle-specific actins (alpha and gamma isoforms), desmin and cytokeratin, and with antibodies against the vascular endothelial marker von Willebrand factor. The squamous cell carcinoma was cytokeratin-positive. Other tumours were cytokeratin-negative and vimentin-positive. Three tumours had scattered individual cells and groups of cells immunoreactive with antibodies against smooth muscle actin and muscle-specific actins; two tumours (a fibrosarcoma and the malignant fibrous histiocytoma) had small numbers of desmin-positive cells. The putative haemangiosarcoma contained two populations of neoplastic cells, von Willebrand factor-positive vascular endothelial cells and smooth muscle actin-positive spindle cells. It was concluded (1) that UVR-induced corneal tumours may be composed of cells derived from resident epithelial cells, immigrant vascular endothelial cells, or fibroblast-like cells of unknown origin, and (2) that such tumours may contain more than one neoplastic cell type.

Differences in the response to oxidative stress and mutant frequency in CD (Sprague-Dawley) and Fisher 344 rats due to an induced inflammatory response.

In this study, the rodent air pouch model was used to examine the production and processing of oxidative DNA damage in two strains of rats commonly used in toxicity testing. An inflammatory response was induced by injecting zymosan A (50 mg) into an air pouch on male CD (Sprague-Dawley [S-D]) and Fisher 344 (F-344) rats, and the animals were then sacrificed at 1, 3, 7, 14, and 28 days (n = 6 per time point per strain). Tissues from the lining of the air pouch were collected for 8-hydroxy-2'-deoxyguanosine (8-OH-dG) analysis and for paraffin embedding. Significant (P < 0.01) increases in 8-OH-dG were observed after 1 day in the DNA from cells lining the air pouch of zymosan A-treated versus control S-D (101.5 +/- 27.1 vs. 23.1 +/- 2. 7 8-OH-dG/dG x 10(5)) and F-344 (51.4 +/- 5.3 vs. 14.4 +/- 0.6 8-OH-dG/dG x 10(5)) rats. By 28 days, 8-OH-dG levels had returned to background in S-D rats, but remained elevated in F-344 rats. The frequency of apoptosis was evaluated using the in situ end-labeling (TUNEL) assay, which revealed that zymosan A-treated S-D rats had a significantly (P < 0.05) higher frequency of apoptosis compared to zymosan A-treated F-344 rats. To examine the potential consequences of these differences in endogenously produced DNA damage and apoptosis, we measured mutations at the hprt locus in fibroblasts of the pouch lining and observed a significant (P < 0.05) increase in the mutant frequency at day 28 in F-344 rats (54.2 +/- 13.6 mutants per 10(6) cells) compared to controls (4.5 +/- 2.0 mutants per 10(6) cells). The mutant frequency was not increased in S-D rats. These data demonstrate that strain differences in the production and processing of oxidative DNA damage due to an inflammatory response may impact the long-term pathologic consequences of chronic inflammation. Environ. Mol. Mutagen. 35:336-342, 2000 Published 2000 Wiley-Liss, Inc.

DNA adduct measurements, cell proliferation and tumor mutation induction in relation to tumor formation in B6C3F1 mice fed coal tar or benzo[a]pyrene.

Coal tar is a complex mixture containing hundreds of compounds, at least 30 of which are polycyclic aromatic hydrocarbons, including the carcinogen benzo[a]pyrene (BaP). Although humans are exposed to complex mixtures on a daily basis, the synergistic or individual effects of components within a mixture on the carcinogenic process remain unclear. We have compared DNA adduct formation and cell proliferation in mice fed coal tar or BaP for 4 weeks with tumor formation in a 2 year chronic feeding study. Additionally, we have analyzed tumor DNA for mutations in the K-ras, H-ras and p53 genes. In the forestomach of mice fed either coal tar or BaP an adduct indicative of BaP was detected, with adduct levels increasing in a dose-responsive manner. K-ras mutations were detected in the forestomach tumors, with the incidence being similar in mice fed coal tar or BaP. These results suggest that the BaP within coal tar is associated with forestomach tumor induction in coal tar-fed mice. DNA adduct levels in the small intestine were not predictive of tumor incidence in this tissue; instead, the tumors appeared to result from compound-induced cell proliferation at high doses of coal tar. K-ras mutations were detected in lung tumors. Since lung tumors were not increased by BaP, coal tar components other than BaP appear to be responsible for the tumors induced in this tissue. H-ras mutations, primarily occurring at codon 61, were the most common mutation observed in liver tumors induced by coal tar. Since this mutation profile is observed in spontaneous hepatic tumors, components in the coal tar may be promoting the expansion of pre-existing lesions.

Interactive effects of methyl-deficiency and dietary restriction on liver cell proliferation and telomerase activity in Fischer 344 rats pretreated with aflatoxin B(1).

The effects of methyl-deficiency and dietary restriction (DR) on hepatic cell proliferation and telomerase activity was studied in male Fischer 344 rats pretreated with aflatoxin B(1) (AFB(1)). Five-week-old rats were gavaged 5 days per week for 3 weeks with AFB(1) (25 microg/rat per day) or solvent (100 microl 75% dimethylsulfoxide). Rats were then divided into four groups. Two groups were fed a methyl-sufficient (MS) diet either ab libitum (AL) or with DR. The other two groups were fed a methyl-deficient (MD) diet either AL or with DR. At 15, 20, and 32 weeks of age, hepatic cell proliferation, telomerase activity, and the number of glutathione S-transferase-P positive (GST-P(+)) foci were determined. DR reduced hepatic cell proliferation, while the MD diet and AFB(1) pretreatment increased cell proliferation. Telomerase activity was decreased by DR and increased by the MD diet and AFB(1) pretreatment. The same trend was observed with GST-P(+) foci: in AFB(1)-pretreated rats, methyl deficiency increased the number of foci, while DR decreased the number. These results are consistent with a role of telomerase in hepatocarcinogenesis.

Stimulated pulmonary cell hyperplasia underlies resistance to alpha-naphthylthiourea.

The rodenticide alpha-naphthylthiourea (ANTU) causes pulmonary edema and pleural effusion that leads to death via pulmonary insufficiency. Rats become resistant to the lethal effect of ANTU if they are first exposed to a small, nonlethal dose of ANTU. Young rats are also resistant to ANTU. The mechanism by which rats develop resistance by a prior, small dose exposure has yet to be determined. Growth factor induced-pulmonary hyperplasia has been demonstrated to attenuate ANTU-induced lung leak. We hypothesized that a small dose of ANTU protects against a large dose through pulmonary cell hyperplasia induced by the protective dose. Furthermore, we hypothesized that this hyperplasia is associated with altered transcription of growth factors. Male Sprague-Dawley rats (175-225 g) were treated with a low dose of ANTU (5 mg ANTU/kg; ANTU(L)) 24 h before challenge with a 100% lethal dose of ANTU (70 mg ANTU/kg; ANTU(H)) resulting in 100% protection against the lethal effect of ANTU(H). ANTU(L) protection against ANTU(H) lasted for 5 days, slowly phased out, all being lost by day 20. Injury was assessed by estimating pulmonary vascular permeability and through histopathological examination. ANTU(H) alone resulted in an increase in pulmonary edema leading to animal death. However, injury was prevented if the rats were first treated with ANTU(L). There was a stimulation of pulmonary cell hyperplasia in the lungs of ANTU(L) treated rats as measured by [3H]-thymidine and bromodeoxyuridine incorporation. Treatment with the antimitotic agent colchicine abolished ANTU(L)-induced resistance to ANTU(H). ANTU resistant rats were also resistant to the lethal effect of paraquat. Paraquat is not taken up by pneumocytes if they are undergoing hyperplasia. ANTU(L) administration resulted in an up regulation of gene transcription for keratinocyte growth factor, transforming growth factor-beta, keratinocyte growth factor receptor and epidermal growth factor receptor as determined through reverse transcription-polymerase chain reaction. A significant increase in transforming growth factor-alpha was not observed. These findings collectively suggest that ANTU(L)-induced pulmonary cell hyperplasia underlies resistance to ANTU(H). Furthermore, the stimulation of hyperplasia may be due to altered growth factor and growth factor receptor expressions.

Temporal changes in tissue repair permit survival of diet-restricted rats from an acute lethal dose of thioacetamide.

Although, diet restriction (DR) has been shown to substantially increase longevity while reducing or delaying the onset of age-related diseases, little is known about the mechanisms underlying the beneficial effects of DR on acute toxic outcomes. An earlier study (S. K. Ramaiah et al., 1998, Toxicol. Appl. Pharmacol. 150, 12-21) revealed that a 35% DR compared to ad libitum (AL) feeding leads to a substantial increase in liver injury of thioacetamide (TA) at a low dose (50 mg/kg, i.p.). Higher liver injury was accompanied by enhanced survival. A prompt and enhanced tissue repair response in DR rats at the low dose (sixfold higher liver injury) occurred, whereas at equitoxic doses (50 mg/kg in DR and 600 mg/kg in AL rats) tissue repair in AL rats was substantially diminished and delayed. The extent of liver injury did not appear to be closely related to the extent of stimulated tissue repair response. The purpose of the present study was to investigate the time course (0-120 h) of liver injury and liver tissue repair at the high dose (600 mg TA/kg, i.p., lethal in AL rats) in AL and DR rats. Male Sprague-Dawley rats (225-275 g) were 35% diet restricted compared to their AL cohorts for 21 days and on day 22 they received a single dose of TA (600 mg/kg, i.p.). Liver injury was assessed by plasma ALT and by histopathological examination of liver sections. Tissue repair was assessed by [3H]thymidine incorporation into hepatonuclear DNA and proliferating cell nuclear antigen (PCNA) immunohistochemistry during 0-120 h after TA injection. In AL-fed rats hepatic necrosis was evident at 12 h, peaked at 60 h, and persisted thereafter until mortality (3 to 6 days). Peak liver injury was approximately twofold higher in DR rats compared to that seen in AL rats. Hepatic necrosis was evident at 36 h, peaked at 48 h, persisted until 96 h, and returned to normal by 120 h. Light microscopy of liver sections revealed progression of hepatic injury in AL rats whereas injury regressed completely leading to recovery of DR rats by 120 h. Progression of injury led to 90% mortality in AL rats vs 30% mortality in DR group. In the surviving AL rats, S-phase DNA synthesis was evident at 60 h, peaked at 72 h, and declined to base level by 120 h, whereas in DR rats S-phase DNA synthesis was evident at 36 h and was consistently higher until 96 h reaching control levels by 120 h. PCNA studies showed a corresponding increase in cells in S and M phase in the AL and DR groups. DR resulted in abolition of the delay in tissue repair associated with the lethal dose of TA in ad libitum rats. Temporal changes and higher tissue repair response in DR rats (earlier and prolonged) are the conduits that allow a significant number of diet restricted rats to escape lethal consequence.

Differential follicle counts as a screen for chemically induced ovarian toxicity in mice: results from continuous breeding bioassays.

Ovaries from National Toxicology Program Reproductive Assessment by Continuous Breeding (RACB) bioassays were used to directly compare differential ovarian follicle counts and reproductive performance for 15 chemicals. Ovaries of 10 animals per group from 16 studies in CD-1 mice and 1 study each in C3H and C57BL/6 mice were sectioned serially at 6 microm. Counts of small, growing, and antral follicles were obtained in every 10th section. For all follicle types, younger mice had more follicles than older mice, and CD-1 mice had more follicles than age-matched animals from either inbred strain. The in-life portion of the RACB protocols demonstrated that 9 of 15 chemicals altered reproductive outcome in one or both sexes of mice, with six agents affecting females (R. E. Morrissey et al., 1989, Fundam. Appl. Toxicol. 13, 747-777). Three of six female toxicants [2,2-bis(bromoethyl)-1,3-propanediol, BPD; ethylene glycol monomethyl ether, EGME; methoxyacetic acid, MAA] significantly decreased counts of small and/or growing follicles by 33 to 92% in CD-1 mice; EGME also reduced follicle counts in the other strains. Follicle counts were decreased in progeny of animals treated with EGME or its active metabolite, MAA. For BPD, reductions in follicle numbers were proportional to dose. In CD-1 mice, female toxicants di-N-hexyl phthalate, propantheline bromide, and tricresyl phosphate reduced reproductive performance but not follicle numbers. Counts were not affected by toxicants for which the susceptible sex could not be determined (bisphenol A, ethylene glycol, oxalic acid). Altered follicle counts without apparent reproductive impairment occurred in CD-1 mice at lower doses of BPD but were not observed for nontoxic chemicals. These data suggest that differential follicle counts (1) are a quantifiable endpoint of ovarian injury in conventional bioassays, and (2) in some instances, may provide a more sensitive indicator of female reproductive toxicity than fertility.

Influence of sampling on the reproducibility of ovarian follicle counts in mouse toxicity studies.

Different ovarian follicle counting procedures were investigated to reduce labor while retaining statistical power. Intact ovaries of untreated CD-1 mice (20/group) from National Toxicology Program Reproductive Assessment by Continuous Breeding (RACB) studies were serially sectioned at 6 microm. Mean numbers of small and growing follicles were used to assess sampling efficiency. In 10 mice per group, comparisons were made between 10% nonrandom samples from every 10th section starting at either the first or sixth section having follicles (approximately 40 sections per ovary). These 10% counts were compared with 5% (20 sections) and 20% (80 sections) nonrandom samples and with 1% (4 sections), 5%, or 10% random samples from the same 10 animals. For two studies, a 10% nonrandom sample was analyzed from 20 mice per group. Follicle counts for each group were comparable regardless of the sampling paradigm. Four to 10 animals provided 90% confidence that a 20% difference in mean counts would be detected. The 1% sample had a larger error term and, thus, slightly reduced statistical power. These data suggest that follicle counts from 1% or 5% random samples may provide a suitable screen for ovarian toxicity.

Antimitotic intervention with colchicine alters the outcome of o-DCB-induced hepatotoxicity in Fischer 344 rats.

Although, hepatotoxic injury of 1,2-dichlorobenzene (o-DCB) is greater in Fischer 344 (F344) as compared to Sprague-Dawley (S-D) rats, this interstrain difference does not transcend into any difference in lethal effects of o-DCB. Interstrain difference in compensatory tissue repair has been suggested as the underlying mechanism for the lack of strain differences in lethality (S.G. Kulkarni, H. Duong, R. Gomila, and H.M. Mehendale, Strain differences in tissue repair response to 1,2-dichlorobenzene. Archives of Toxicology 1996; 70: 714-723). If higher tissue repair in F344 rats compensates for more severe liver injury, then antimitotic intervention after infliction of o-DCB-induced liver injury should lead to lethality in F344 rats. Colchicine (CLC, 1 mg/kg) functions as an effective antimitotic agent and does not cause any side effects apart from suppressing cellular proliferation. Two groups of male F344 rats (160-190 g) received a single dose of 0.6 ml o-DCB/kg: 30 h later one group of rats received CLC (1 mg/kg; i.p.) and the other received distilled water (1 ml/kg; i.p.). Liver injury was assessed by measuring plasma ALT and SDH activity, liver histopathology, and liver regeneration was estimated by [3H]thymidine incorporation into hepatonuclear DNA and proliferating cell nuclear antigen (PCNA) assay in both groups. Similar liver injury was noted in both the o-DCB + vehicle and o-DCB + CLC treated F344 rats at 36 h indicating that CLC does not interfere with the uptake, bioactivation and causation of injury by o-DCB. S-phase synthesis which occurred at 36 h in the o-DCB + vehicle group was blocked in the o-DCB + CLC group. CLC administration 6 h prior to S-phase stimulation selectively abolished S-phase stimulation at 36 h, and led to 50% lethality. Since the effect of CLC antimitosis was transient, S-phase synthesis occurring at 48 h was not blocked and was sustained up to 72 h thereby allowing the other 50% of rats to overcome liver injury induced by o-DCB and survive the lethal outcome. These findings suggest that a significantly higher rate of compensatory tissue repair in F344 rats enables them to overcome more severe liver injury inflicted by o-DCB.

Genome-linked toxic responses to dietary iron overload.

Genome-related differences to Fe overload between and within rodent species were evaluated in the present study. Male B6C3F1 mice, yellow and black C5YSF1 mice, and Fischer 344 (F344) rats were fed AIN-76A diets containing 35 (control), 1,500, 3,500, 5,000, or 10,000 micrograms carbonyl Fe/g for 12 wk. No effects on body weight gain were observed in the B6C3F1 and black C5YSF1 mice, whereas at all doses of Fe above the control, weight gain was reduced in yellow C5YSF1 mice and F344 rats. At the 10,000 micrograms Fe/g dose, 9 of 12 rats died, but there was no mortality among the mice. In all animals, there was a dose-related increase in liver nonheme Fe, and the Fe was stored in hepatocytes predominantly in the periportal region. There was significant hypertrophy of the hepatocytes in both B6C3F1 mice and F344 rats fed the 10,000 micrograms Fe/g diet. PCNA assays showed significant stimulatory effects of the high dose of Fe on hepatocyte proliferation in the F344 rats and the C5YSF1 mice but not in the B6C3F1 mice. In the rat, there was pancreatic atrophy with loss of both endocrine and exocrine tissue. Morphometric evaluation of pancreas showed fewer beta cells in B6C3F1 and yellow C5YSF1 mice but not in the black C5YSF1 mice. There were fewer islets in the yellow C5YSF1 mice, and total and mean islet areas were smaller than in the control mice. Rats in the 10,000 micrograms Fe/g dose group had markedly exacerbated dose-dependent nephropathy and changes in glomerular and tubular epithelium associated with Fe accumulation. The rats also showed degeneration of the germinal epithelium of the testis, formation of multinucleated giant cells, and lack of mature sperm.

Glaucoma in food-restricted and ad libitum-fed DBA/2NNia mice.

We allocated 110 DBA/2NNia mice of either sex to one of two feeding regimens: ad libitum (AL) or food restriction (FR) to 60% of the amount consumed by the AL group. The mice were examined at 3, 6, 9, 12, 18, and 24 months (at 3 months, only AL mice were examined). During the remaining periods approximately equal numbers (n = 10) of mice of both sexes and diet groups were examined. Peripheral anterior synechia was the first glaucoma-associated lesion observed and was present in 8 of 10 AL female mice, 6 of 10 AL males, and 1 FR male at 6 months. At 9 months peripheral anterior synechia was present in all AL females and was accompanied by depletion of retinal ganglion cells and degeneration of the optic nerves and optic tracts. Ninety percent of the eyes in the AL males also had peripheral anterior synechia at 9 months, but ganglion cell depletion and optic nerve degeneration were not observed as frequently. Neovascular membranes in the iridocorneal angle, a component of peripheral anterior synechia, were first observed at 9 months in approximately 55% of the globes of the AL mice and 5% of the FR mice. This was a major difference in the microscopic features of synechia between the diet groups and resulted in increased severity of synechia in the AL mice compared with their FR cohorts. Degeneration of the optic nerves and tracts was characterized by atrophy, astrogliosis, increase in cellularity, fragmentation of axons, and loss of myelin. Glaucoma in the FR mice of both sexes was less severe than in their AL counterparts. The most severely affected were AL females, followed by FR females, AL males, and FR males. Food restriction reduced the incidence and severity of the ocular lesions in females at all periods. The primary benefit of FR in males occurred during the 6- and 9-month periods when the incidence and severity of the glaucoma-related lesions were reduced; in the succeeding months the major benefit was minimal reduction of the severity of the lesions.