Genistein and ethinyl estradiol dietary exposure in multigenerational and chronic studies induce similar proliferative lesions in mammary gland of male Sprague-Dawley rats.

Genistein and ethinyl estradiol (EE(2)) were examined in multigenerational reproductive and 2-yr chronic toxicity studies with different exposure durations across generations F(0) through F(4). Sprague-Dawley rats were exposed to genistein (0, 5, 100, or 500 ppm) or EE(2) (0, 2, 10, or 50 ppb). Effects in the male mammary gland are described here. In the multigeneration studies, mammary hyperplasia was induced by both compounds; the chronic studies had a lower incidence, without proportionate neoplasia. Sexual dimorphism (predominant tubuloalveolar growth in females and lobuloalveolar in males) was retained without feminization in high dose genistein or EE(2). In the continuously exposed generations, mammary hyperplasia was sustained but not amplified, appeared morphologically similar across all generations, and was not carried over into unexposed offspring of previously exposed generations. The hyperplasia in male rats was similar whether induced by genistein or EE(2). Results substantiate and extend previous reports that mammary gland hyperplasia in the male rat is one of the most sensitive markers of estrogenic endocrine disruption.

Overlapping but distinct effects of genistein and ethinyl estradiol (EE(2)) in female Sprague-Dawley rats in multigenerational reproductive and chronic toxicity studies.

Genistein and ethinyl estradiol (EE(2)) were examined in multigenerational reproductive and chronic toxicity studies that had different treatment intervals among generations. Sprague-Dawley rats received genistein (0, 5, 100, or 500 ppm) or EE(2) (0, 2, 10, or 50 ppb) in a low phytoestrogen diet. Nonneoplastic effects in females are summarized here. Genistein at 500 ppm and EE(2) at 50 ppb produced similar effects in continuously exposed rats, including decreased body weights, accelerated vaginal opening, and altered estrous cycles in young animals. At the high dose, anogenital distance was subtly affected by both compounds, and a reduction in litter size was evident in genistein-treated animals. Genistein at 500 ppm induced an early onset of aberrant cycles relative to controls in the chronic studies. EE(2) significantly increased the incidence of uterine lesions (atypical focal hyperplasia and squamous metaplasia). These compound-specific effects appeared to be enhanced in the offspring of prior exposed generations.

Calpain released from dying hepatocytes mediates progression of acute liver injury induced by model hepatotoxicants.

Liver injury is known to progress even after the hepatotoxicant is long gone and the mechanisms of progressive injury are not understood. We tested the hypothesis that hydrolytic enzymes such as calpain, released from dying hepatocytes, destroy the surrounding cells causing progression of injury. Calpain inhibitor, N-CBZ-VAL-PHE-methyl ester (CBZ), administered 1 h after a toxic but nonlethal dose of CCl(4) (2 ml/kg, ip) to male Sprague Dawley rats substantially mitigated the progression of liver injury (6 to 48 h) and also led to 75% protection against CCl(4)-induced lethality following a lethal dose (LD75) of CCl(4) (3 ml/kg). Calpain leakage in plasma and in the perinecrotic areas increased until 48 h and decreased from 72 h onward paralleling progression and regression of liver injury, respectively, after CCl(4) treatment. Mitigation of progressive injury was accompanied by substantially low calpain in perinecrotic areas and in plasma after CBZ treatment. Normal hepatocytes incubated with the plasma collected from CCl(4)-treated rats (collected at 12 h when most of the CCl(4) is eliminated) resulted in extensive cell death prevented by CBZ. Cell-impermeable calpain inhibitor E64 also protected against progression of CCl(4)-induced liver injury, thereby confirming the role of released calpain in progression of liver injury. Following CCl(4) treatment, calpain-specific breakdown of alpha-fodrin increased, while it was negligible in rats receiving CBZ after CCl(4). Hepatocyte cell death in incubations containing calpain was completely prevented by CBZ. Eighty percent of Swiss Webster mice receiving a lethal dose (LD80) of acetaminophen (600 mg/kg, ip) survived if CBZ was administered 1 h after acetaminophen, suggesting that calpain-mediated progression of liver injury is neither species nor chemical specific. These findings suggest the role of calpain in progression of liver injury.

Activation of PPAR-alpha in streptozotocin-induced diabetes is essential for resistance against acetaminophen toxicity.

Diabetic (DB) mice exhibit significant resistance to hepatotoxicants. The role of peroxisome proliferator receptor (PPAR)-alpha activation in diabetes, in protection against lethal acetaminophen (APAP) challenge, was investigated. Upon treatment with APAP (600 mg/kg, i.p., a LD100 dose in wild-type [WT] non-DB mice), WT-DB mice showed only 30% mortality and 40% less liver injury as measured by alanine aminotransferase and histopathology. In contrast, diabetes in PPAR knockout (PPAR-alpha-/-) mice failed to protect against APAP toxicity, suggesting the importance of PPAR-alpha in diabetes-induced protection. S-phase DNA synthesis and PCNA immunohistochemical staining after injury showed early and robust tissue repair in WT-DB mice, but not in the PPAR-alpha-/--DB mice. Microarray analyses were performed on livers from non-DB and DB (WT and PPAR-alpha-/-) mice at 0 and 12 h after APAP. Microarray data were confirmed via real-time polymerase chain reaction analysis of several genes, including stress response, immediate early genes, DNA damage, heat shock proteins, and cell cycle regulators, followed by Western analyses of selected proteins. Gel shift assays revealed higher activation of nuclear factor-kappaB in WT-DB mice after APAP treatment. These findings suggest PPAR-alpha activation as a hepatoprotective adaptive response mediating protection against APAP in diabetes.

Two-generation reproductive study in mink fed diisopropyl methylphosphonate (DIMP).

Two generations of "Ranch Wild" mink (Mustela vison) were fed the organophosphate diisopropyl methylphosphonate (DIMP) at 0, 150, 450, or 1250ppm, to determine potential toxicity to the dams. Chemical, hematologic, necropsy, and microscopic examinations were performed on all parental animals and representative kits. The F0 and F1 dams had 3.4 and 4.6% mortality, respectively, distributed among all groups and not attributed to DIMP exposure. Adverse effects were mild and limited to the highest dose group. Plasma cholinesterase was reduced 40% (F0) and 31% (F1), as was whole blood cholinesterase (16 and 8.5%). Heinz bodies were present in 2.8% (F0) and 1.3% (F1) of erythrocytes. The erythrocyte count was reduced 6.3% in the F0. Reproductive efficiency was not affected. The mink were not uniquely susceptible to DIMP, relative to the literature on other species. The no observed adverse effect level (NOAEL), based on the 450ppm group of F1 females, was 56.5mg DIMP/kgBW per day; the lowest observed adverse effect level (LOAEL) was 329.5mg DIMP/kgBW per day.

Role of tissue repair in survival from s-(1,2-dichlorovinyl)-L-cysteine-induced acute renal tubular necrosis in the mouse.

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), a model nephrotoxicant in mice, causes acute tubular necrosis and death at high doses. Our earlier studies revealed that renal tissue repair was critical for survival in mice with DCVC nephrotoxicity. The objective of this study was to investigate if increasing renal tissue repair could protect mice from the lethal outcome of DCVC. Male Swiss Webster (SW) mice were administered a low dose of DCVC (15 mg/kg, ip) 72 h before injection of a normally lethal dose of DCVC (75 mg/kg, ip); this resulted in 100% protection against the lethal effect of DCVC. Because DCVC caused approximately two fold decrease in cytosolic and mitochondrial beta-lyase activity, the possibility that DCVC protection may be caused by decreased bioactivation was examined. Mercuric chloride (HgCl2, 6 mg/kg), a nephrotoxicant with no effect on beta-lyase activity, was administered 96 h before a lethal dose of DCVC. This also resulted in 100% protection from the lethal effect of DCVC. In both studies total glutathione was unchanged at any time after the lethal dose of DCVC was administered, obviating the role of glutathione in protection. In both cases the augmented and sustained tissue repair induced by priming dose and documented by 3H-thymidine pulse labeling and immunocytochemistry for proliferating cell nuclear antigen resulted in 100% survival in spite of the extensive renal injury. These findings suggest that stimulation of renal tubular repair by the priming dose, through augmented cell division, and the resistance of new cells to mechanisms of progression of injury, underlies auto- and heteroprotection against DCVC. The molecular mechanisms may have potential application in pharmacotherapeutic intervention for treatment of acute renal failure.

Renal injury and repair following S-1, 2 dichlorovinyl-L-cysteine administration to mice.

S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a metabolite of a common environmental contaminant, trichloroethylene, is a selective proximal tubular nephrotoxicant. The objective of our study was to examine the dose-response relationship of renal injury and repair following DCVC administration. Male Swiss-Webster mice were injected with DCVC [15, 30, or 75 mg/kg ip in distilled water (10 ml/kg)] and the extent of nephrotoxicity and tissue repair was assessed over a 14-day period. The renal injury due to the low and medium doses of DCVC peaked at 36 and 72 h after dosing, respectively, and then regressed over time due to a timely and adequate tissue repair response. At the highest dose tissue repair was inhibited, thereby causing progression of renal injury, which led to acute renal failure and death of the mice. The possibility that compromised tissue repair was a result of the extensive nephrotoxic injury attendant to the high dose of DCVC was investigated via an equinephrotoxicity study in which separate groups of mice received 40 (LD40) and 75 (LD90) mg DCVC/kg, respectively. Bioactivation-based renal proximal tubular injury measured in these two groups over a time course was identical but there was a marked difference in mortality due to an early and robust tissue repair in the first group relative to the second group. These results support the concept that quantitative evaluation of renal tissue repair in parallel with injury is useful in the assessment of the likely toxic outcome associated with exposure to nephrotoxic drugs and toxicants.

Streptozotocin-induced diabetic mice are resistant to lethal effects of thioacetamide hepatotoxicity.

The effect of Type 1 diabetes on the toxicity of thioacetamide was investigated in a murine model. In streptozotocin-induced diabetic C57BL6 mice a LD90 dose of thioacetamide (1000 mg/kg, ip in saline) caused only 10% mortality. Alanine aminotransferase activity revealed approximately 2.7-fold less liver injury in the diabetic (DB) mice compared to the non-DB controls, at 36 h after thioacetamide (TA) administration, which was confirmed via histopathological analysis. HPLC analyses revealed lower plasma t(1/2) of TA in the DB mice. Covalent binding of [(14)C]TA to liver tissue was lower in the DB mice, suggesting lower bioactivation of TA. Compensatory hepatic S-phase stimulation as assessed by [(3)H]thymidine incorporation occurred much earlier and was substantially higher in the DB mice compared to the non-DB cohorts. Morphometric analysis of cells in various phases of cell division assessed via immunohistochemical staining for proliferating cell nuclear antigen revealed more cells in G(1), S, G(2), and M phases in the DB mice, indicating robust tissue repair in concordance with the findings of [(3)H]thymidine pulse labeling studies. The importance of tissue repair in the resistance of DB mice was further investigated by blocking cell division in the DB mice by colchicine (1 mg/kg, ip) at 40 h after TA administration, well after the bioactivation of TA. Antimitotic action of colchicine, confirmed by decreased S-phase stimulation, led to progression of liver injury and increased mortality in DB mice. These findings suggest that lower bioactivation of TA and early onset of liver tissue repair are the pivotal underpinnings for the resistance of DB mice.

Type 1 diabetic mice are protected from acetaminophen hepatotoxicity.

Streptozotocin (STZ)-induced diabetic (DB) mice challenged with single ordinarily lethal doses of acetaminophen (APAP), carbon tetrachloride (CCl4), or bromobenzene (BB) were resistant to all three hepatotoxicants. Mechanisms of protection against APAP hepatotoxicity were investigated. Plasma alanine aminotransferase, aspartate aminotransferase, and liver histopathology revealed significantly lower hepatic injury in DB mice after APAP administration. HPLC analysis of plasma and urine revealed lower plasma t1/2, increased volume of distribution (Vd), and increased plasma clearance (CLp) of APAP in the DB mice and no difference in APAP-glucuronide, a major metabolite in mice. Interestingly, covalent binding of 14C-labeled APAP to liver target proteins; arylation of APAP to 58, 56, and 44 kDa acetaminophen binding proteins (ABPs); and glutathione (GSH) depletion in the liver did not differ between nondiabetic (non-DB) and DB mice in spite of downregulated hepatic microsomal CYP2E1 and 1A2 proteins in the DB mice, known to be involved in bioactivation of APAP. Compensatory cell division measured via 3H-thymidine pulse labeling and immunohistochemical staining for proliferating cell nuclear antigen (PCNA) indicated earlier onset of S-phase in the DB mice after exposure to APAP. Antimitotic intervention of liver cell division by colchicine (CLC) after administration of APAP led to significantly higher mortality in the DB mice suggesting a pivotal role of liver cell division and tissue repair in the protection afforded by diabetes. In conclusion, the resistance of DB mice against hepatotoxic and lethal effects of APAP appears to be mediated by a combination of enhanced APAP clearance and robust compensatory tissue repair.

Mechanisms of increased liver tissue repair and survival in diet-restricted rats treated with equitoxic doses of thioacetamide.

Moderate dietary or caloric restriction (DR) modulates animal physiology in a beneficial fashion. Previously, we have reported an equitoxic dose experiment where liver injury in DR male Sprague-Dawley rats exposed to a low dose of thioacetamide (TA, 50 mg/kg) was similar to that observed in ad libitum fed (AL) rats exposed to a 12-fold higher dose (600 mg/kg). Paradoxically, the AL rats experienced 90% mortality while all of the DR rats, with the same amount of initial bioactivation-mediated liver injury, survived. The protection observed in the DR rats was due to efficient compensatory liver tissue repair, which was delayed and attenuated in the AL rats, leading to progression of liver injury. The objective of the present study was to investigate the molecular mechanisms of the enhanced tissue repair in the DR rats upon equitoxic challenge with TA. Promitogenic mechanisms and mediators such as proinflammatory cytokines (TNF-alpha and IL-6), growth factors (TGF-alpha and HGF), and inducible nitric oxide synthase (iNOS) were estimated over a time course after equitoxic challenge (50 mg/kg to DR vs. 600 mg/kg to AL rats). Except for TNF-alpha, all other molecules were expressed earlier and in greater amount in the DR rats. IL-6 was 10-fold greater and peaked 12 h earlier; HGF also peaked 12 h sooner in the DR rats, when it was 2.5-fold greater than the value in the AL rats. TGF-alpha expression in livers of DR rats increased after TA administration and peaked at 24 h. In the AL rats, it was lower and peaked at 36 h. Diet restriction alone induced iNOS 2-fold in the DR rats and remained elevated until 12 h after TA administration, then declined thereafter. The lower iNOS activity in the AL rats further decreased after TA injection. DR rats exhibited higher apoptosis after thioacetamide administration, which further increased the efficiency of tissue repair. Taken together, these data indicate that even though the liver injury is near equal in AL and DR rats, sluggish signal transduction leads to delayed liver regeneration, progression of liver injury, and death in the AL rats. The equitoxic dose experiment indicates that stimulation of tissue repair is independent of the extent of initial liver injury and is governed by physiology of diet restriction. DR stimulates promitogenic signaling leading to a quick and timely response upon liver injury, arrest of progressive injury on one hand, and recovery from injury on the other, paving the way for survival of the DR rats.

Upregulated promitogenic signaling via cytokines and growth factors: potential mechanism of robust liver tissue repair in calorie-restricted rats upon toxic challenge.

Previously we reported that moderate calorie restriction or diet restriction (DR, calories reduced by 35% for 21 days) in male Sprague-Dawley rats protects from a lethal dose of thioacetamide (TA). DR rats had 70% survival compared with 10% in rats fed ad libitum (AL) because of timely and adequate compensatory liver cell division and tissue repair in the DR rats. Further investigation of the mechanisms indicate that enhanced promitogenic signaling plays a critical role in this stimulated tissue repair. Expression of stimulators of promitogenic signaling interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), hepatocyte growth factor (HGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGFR) were studied during liver tissue repair after TA-induced liver injury. Plasma IL-6 was significantly higher in the DR rats, with 6-fold higher expression at 48 h after TA administration. Immunohistochemical localization revealed significantly higher expression of IL-6 in the hepatic sinusoidal endothelium of DR rats. Expression of TGF-alpha and HGF was consistently higher in the livers of DR rats from 36 to 72 h. EGFR, which serves as a receptor for TGF-alpha, was higher in DR rats before TA administration and remained higher till 48 h after TA intoxication. DR-induced 2-fold increase in hepatic iNOS activity is consistent with early cell division in DR rats after TA challenge. These data suggest that the reason behind the higher liver tissue repair after TA-induced hepatotoxicity in DR rats is timely and higher expression of the growth stimulatory cytokines and growth factors. It appears that the physiological effects of DR make the liver cells vigilant and prime the liver tissue promptly for liver regeneration through promitogenic signaling upon toxic challenge.

Tumorigenicity of chloral hydrate, trichloroacetic acid, trichloroethanol, malondialdehyde, 4-hydroxy-2-nonenal, crotonaldehyde, and acrolein in the B6C3F(1) neonatal mouse.

The tumorigenicity of chloral hydrate (CH), trichloroacetic acid (TCA), trichloroethanol (TCE), malondialdehyde (MDA), crotonaldehyde, acrolein, and 4-hydroxy-2-nonenal (HNE) was tested in the B6C3F(1) neonatal mouse. Mice were administered i.p. injections of CH (1000, 2000, 2500, and 5000 nmol per animal), TCA (1000 and 2000 nmol), TCE (1000 and 2000 nmol), MDA (1500 and 3000 nmol), crotonaldehyde (1500 and 3000 nmol), acrolein (75 and 150 nmol), and HNE (750 and 1500 nmol) at 8 and 15 days of age. At 12 months, only male mice treated with the positive control chemicals, 4-aminobiphenyl (500 and 1000 nmol) and benzo[a]pyrene (150 and 300 nmol), had incidences of tumors in the liver significantly higher than the solvent control. Additional male mice were dosed as described above and their livers were excised at 24, 48 h, and 7 days after the final dose. Liver DNA was isolated and analyzed by 32P-postlabeling/high-performance liquid chromatography (HPLC) and HPLC/electrochemical detection for MDA-derived adduct (M(1)G) and 8-oxo-2'-deoxyguanosine (8-OHdG) formation, respectively. At 24 and 48 h after the final dose, CH- and TCA-treated mice exhibited significantly higher M(1)G levels than the controls. 8-OHdG formation was also induced by CH, TCA, and MDA. These results suggest that under these experimental conditions the B6C3F(1) neonatal mouse is not sensitive to carcinogens that induce an increase in endogenous DNA adduct formation through lipid peroxidation or oxidative stress.

An immunohistochemical label to facilitate counting of ovarian follicles.

U.S. and internationally harmonized Health Effects Test Guidelines for Reproduction and Fertility Effects include enumeration of primordial and developing ovarian follicles as endpoints of safety tests, and the number of these structures is also of interest for other aspects of reproductive biology. Performing the counts microscopically on representative hematoxylin and eosin (H&E)-stained sections of ovary is tedious and error-prone. The ability to mark oocyte nuclei distinctly with an antibody significantly increases speed and accuracy of counting. We have identified a rabbit polyclonal antibody directed against a synthetic 14-amino acid sequence from human cytochrome P-450 1B1 (CYP1B1) that unequivocally marks rodent oocyte nuclei, in addition to nuclei of some ovarian granulosa and theca cells. Follicles of all degrees of maturity are easily distinguished from ovarian background; ability to detect and identify primordial follicles is particularly enhanced. High-contrast and high-resolution labeling was achieved with routine immunohistochemical procedures using an avidin-biotin-peroxidase method on rat and mouse tissues fixed in 10% neutral buffered formalin.

Carcinogenicity and mechanism of action of fumonisin B1: a mycotoxin produced by Fusarium moniliforme (= F. verticillioides).

Fumonisins are fungal metabolites and suspected human carcinogens. They inhibit ceramide synthase in vitro, enhance tumor necrosis factor alpha (TNFalpha) production, and cause apoptosis. Fumonisin B1 (FB1) was fed to rats and mice for 2 years or, in separate studies, given to rats or mice for up to 4 weeks. Kidney tubule adenomas and carcinomas were found in male rats fed > or = 50 ppm, whereas liver adenomas and carcinomas were found in female mice fed > or = 50 ppm for 2 years. In the short-term studies, increases in tissue concentration of the ceramide synthase substrate sphinganine (Sa) and the Sa to sphingosine (So) ratio were correlated with apoptosis. Further, hepatotoxicity was ameliorated in mice lacking either the TNFR1 or the TNFR2 TNFalpha receptors. Thus, FB1 was carcinogenic to rodents and thefindings support the hypothesis that disrupted sphingolipid metabolism and TNFalpha play important roles in its mode of action.

Effect of inhibitors of nitric oxide synthase on acetaminophen-induced hepatotoxicity in mice.

We recently reported that following a toxic dose of acetaminophen to mice, tyrosine nitration occurs in the protein of cells that become necrotic. Nitration of tyrosine is by peroxynitrite, a species formed from nitric oxide (NO) and superoxide. In this manuscript we studied the effects of the NO synthase inhibitors N-monomethyl-l-arginine (l-NMMA), N-nitro-l-arginine methyl ester (NAME), l-N-(1-iminoethyl)lysine (l-NIL), and aminoguanidine on acetaminophen hepatotoxicity. Acetaminophen (300 mg/kg) increased serum nitrate/nitrite and alanine aminotransferase (ALT) levels, indicating increased NO synthesis and liver necrosis, respectively. None of the NO synthase inhibitors reduced serum ALT levels. In fact, l-NMMA, l-NIL, and aminoguanidine significantly augmented acetaminophen hepatotoxicity at 4 h. A detailed time course indicated that aminoguanidine (15 mg/kg at 0 h and 15 mg/kg at 2 h) significantly increased serum ALT levels over that for acetaminophen alone at 2 and 4 h; however, at 6 and 8 h serum ALT levels in the two groups were identical. At 2 h following acetaminophen plus aminoguanidine NO synthesis was significantly increased; however, at 4, 6, and 8 h NO synthesis was significantly decreased. Aminoguanidine also decreased acetaminophen-induced nitration of tyrosine. Acetaminophen alone did not induce lipid peroxidation, but acetaminophen plus aminoguanidine significantly increased hepatic lipid peroxidation (malondialdehyde levels) at 2, 4, and 6 h. These data are consistent with NO having a critical role in controlling superoxide-mediated lipid peroxidation in acetaminophen hepatotoxicity. Thus, acetaminophen hepatotoxicity may be mediated by either lipid peroxidation or by peroxynitrite.

Dietary restriction alters cell proliferation in rats: an immunohistochemical study by labeling proliferating cell nuclear antigen.

Dietary restriction (DR) delays the onset of aging and lowers the incidence of both spontaneous and chemically induced cancers. The inhibition of cell proliferation has been suggested as a possible mechanism for this effect. We examined the effect of DR on cell proliferation in duodenum, forestomach, glandular stomach, and liver tissues of male Fischer 344 rats receiving 60% of the control feed intake for 24 months starting at 16 weeks of age. Rats were sacrificed, when 28 months old. Tissues were collected, histologically prepared, and stained immunohistochemically for proliferating cell nuclear antigen (PCNA). The PCNA-stained nuclei are detected in different phases of the cell cycle. A minimum sample of 2000 cells was counted in liver. The percentage of labeled S-phase cells per total cells counted was used as the labeling index for liver. The number of labeled S-phase epithelial cells per 1.1 mm of basement membrane or muscularis mucosa was used as the labeling index for duodenum, forestomach, and glandular stomach. Cell proliferation in glandular stomach and liver tissues was inhibited in rats DR for 24 months; however, cell proliferation in duodenum and forestomach mucosal tissues was unexpectedly enhanced by DR. These results indicated that while DR inhibits cell proliferation in tissues of rats, it is tissue-dependent. The decreased rate of cell division by DR in the designated tissues could be implicated in lowering the conversion of endogenous DNA damage or lesions to mutation and cancer.