The intentional adulteration of pet food with melamine and derivatives, including cyanuric acid, has been implicated in the kidney failure and death of a large number of cats and dogs in the United States. Although individually these compounds present low toxicity, coexposure can lead to the formation of melamine cyanurate crystals in the nephrons and eventual kidney failure. To determine the dose-response for nephrotoxicity upon coadministration of melamine and cyanuric acid, groups of male and female F344 rats (six animals per sex per group) were fed 0 (control), 7, 23, 69, 229, or 694 ppm of both melamine and cyanuric acid; 1388 ppm melamine; or 1388 ppm cyanuric acid in the diet for 7 days. No toxicity was observed in the rats exposed to the individual compounds, whereas anorexia and a statistically significant increase in blood urea nitrogen and serum creatinine levels was observed in the animals treated with 229 and 694 ppm melamine and cyanuric acid. The kidneys of these animals were grossly enlarged and pale yellow. Large numbers of crystalline structures deposited in the tubules were seen on sections in kidneys from all rats in these treatment groups. No significant changes were detected in the remaining treatment groups exposed to both melamine and cyanuric acid. In the melamine-only treatment group, 5 of 12 rats had scattered crystals present in renal tubules when examined by wet mount. These were not observed by histopathology. The observed adverse effect level (8.6 mg/kg bw [body weight]/day) and benchmark dose modeling data (8.4-10.9 mg/kg bw/day) determined in this study suggest that the tolerable daily intake values derived from studies conducted with melamine alone may underestimate the risk from coexposures to melamine and cyanuric acid.

A study was undertaken to determine whether alterations in the gene expression or overt histological signs of neurotoxicity in selected regions of the forebrain might occur from acrylamide exposure via drinking water. Gene expression at the mRNA level was evaluated by cDNA array and/or RT-PCR analysis in the striatum, substantia nigra and parietal cortex of rat after a 2-week acrylamide exposure. The highest dose tested (maximally tolerated) of approximately 44 mg/kg/day resulted in a significant decreased body weight, sluggishness, and locomotor activity reduction. These physiological effects were not accompanied by prominent changes in gene expression in the forebrain. All the expression changes seen in the 1200 genes that were evaluated in the three brain regions were < or =1.5-fold, and most not significant. Very few, if any, statistically significant changes were seen in mRNA levels of the more than 50 genes directly related to the cholinergic, noradrenergic, GABAergic or glutamatergic neurotransmitter systems in the striatum, substantia nigra or parietal cortex. All the expression changes observed in genes related to dopaminergic function were less than 1.5-fold and not statistically significant and the 5HT1b receptor was the only serotonin-related gene affected. Therefore, gene expression changes were few and modest in basal ganglia and sensory cortex at a time when the behavioral manifestations of acrylamide toxicity had become prominent. No histological evidence of axonal, dendritic or neuronal cell body damage was found in the forebrain due to the acrylamide exposure. As well, microglial activation was not present. These findings are consistent with the absence of expression changes in genes related to changes in neuroinflammation or neurotoxicity. Over all, these data suggest that oral ingestion of acrylamide in drinking water or food, even at maximally tolerable levels, induced neither marked changes in gene expression nor neurotoxicity in the motor and somatosensory areas of the central nervous system.

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.

The p53 codon 270 CGT to TGT mutation was investigated as a biomarker of sunlight-induced mutagenesis and carcinogenesis. The relationship between tumor development and abundance of this hotspot mutation was analyzed in mouse skin tumors induced by chronic exposure to simulated solar light (SSL). The 24 tumors analyzed had similar growth kinetics, with an average doubling time of similar to 16.4 d. Levels of the p53 codon 270 mutation were quantified in the 24 mouse skin tumors using allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). All tumors contained measurable amounts of the mutation. The p53 codon 270 CGT to TGT mutant fraction (MF) ranged from 2.29 x 10(-3) to 9.42 X 10(-2), with 3.26 x 10(-2) as the median. These p53 MF measurements are lower than expected for an initiating mutation involved in the development of tumors of monoclonal origin. There was no evidence of a correlation between p53 codon 270 MF and either tumor area or an estimate of tumor cell number. Thus, the data do not support the idea that p53 mutation accumulates linearly during tumor development. To investigate how p53 mutation was distributed within tumors, 19 needle biopsies from seven different tumors were analyzed by ACB-PCR. This analysis demonstrated that p53 codon 270 mutation is heterogeneously distributed within tumors. The long-term goal of this research is to combine morphological and p53 MF measurements from tissues corresponding to the various stages of tumor development, in order to derive mathematical models relating the p53 codon 270 mutation to the development of SSL-induced skin tumors. Published 2008 Wiley-Liss, Inc.

Uveal melanoma (UM) is uncommon among wild type mice. Efforts to develop transgenic mice to study this disease have resulted in pigmented tumors derived from the retinal pigment epithelium (RPE) or mixed tumors of RPE and UM complicating the study of UM specifically. Reported here are two early stage intraocular amelanotic melanomas discovered in 2 Tyr-HRAS+ Ink4a/Arf heterozygous (1 normal CKDN2A allele) transgenic FVB/n mice. These tumors were morphologically and immunohistochemically similar to spontaneous UM recently reported in the Ink4a/Arf homozygous (CKDN2A knockout) parent strain. The tumors originated in the posterior uveal tract. The neoplasms were comprised of bundles of spindle-shaped melanocytes admixed with some epithelioid cells. Tumors were immunohistochemically positive for neuron-specific enolase, S-100, pan-ras, but negative for cytokeratin and Melan-A. The development of early lenticular opacity and bilateral cataracts is a consistent phenotype of transgenic mice in which the retinoblastoma signaling pathway has been disrupted. Lenticular opacity and cataracts are rarely observed clinically in Tyr-HRAS+ Ink4a/Arf heterozygotes, rendering this strain suitable for ophthalmoscopy. Consequently, Tyr-HRAS+ Ink4a/Arf heterozygotes provide practical advantages, compared to the cataract-prone CKDN2A knockout strains, for real-time ophthalomoscopic detection and monitoring of UM while developing chemotherapeutic regimens and other research to understand the biology of UM.

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.

Breast cancer is the most common malignancy in women continuing to rise worldwide. Breast cancer emerges through a multi-step process, encompassing progressive changes from a normal cell to hyperplasia (with and without atypia), carcinoma in situ, invasive carcinoma, and metastasis. In the current study, we analyzed the morphological changes and alterations of DNA methylation, histone methylation and microRNA expression during estradiol-17β (E2)-induced mammary carcinogenesis in female August Copenhagen Irish (ACI) rats. E2-induced breast carcinogenesis in ACI rats provides a physiologically relevant and genetically defined animal model for studying human sporadic breast cancer. The pattern of morphological changes in mammary glands during E2-induced carcinogenesis was characterized by transition from normal appearing alveolar and ductular hyperplasia to focal hyperplastic areas of atypical glands and ducts accompanied by a rapid and sustained loss of global DNA methylation, LINE-1 hypomethylation, loss of histone H3 lysine 9 and histone H4 lysine 20 trimethylation, and altered microRNAs expression. More importantly, these alterations in the mammary tissue occurred after 6 weeks of E2-treatment, whereas the atypical hyperplasia, which represents a putative precursor lesion to mammary carcinoma in this model, was detected only after 12 weeks of exposure, demonstrating clearly that these events are directly associated with the effects of E2 and are not a consequence of the preexisting preneoplastic lesions. The results of this study show that deregulation of cellular epigenetic processes plays a crucial role in the mechanism of E2-induced mammary carcinogenesis in ACI rats, especially in the tumor initiation process.

Topical exposure to nanoscale materials is likely from a variety of sources including sunscreens and cosmetics. Because the in vivo disposition of nanoscale materials is not well understood, we have evaluated the distribution of quantum dots (QDs) following intradermal injection into female SKH-1 hairless mice as a model system for determining tissue localization following intradermal infiltration. The QD (CdSe core, CdS capped, poly[ethylene glycol] coated, 37 nm diameter, 621 nm fluorescence emission) were injected intradermally (ID) on the right dorsal flank. Within minutes following intradermal injection, the highly UV fluorescent QD could be observed moving from the injection sites apparently through the lymphatic duct system to regional lymph nodes. Residual fluorescent QD remained at the site of injection until necropsy at 24 h. Quantification of cadmium and selenium levels after 0, 4, 8, 12, or 24 h in multiple tissues, using inductively coupled plasma mass spectrometry (ICP-MS), showed a time-dependent loss of cadmium from the injection site, and accumulation in the liver, regional draining lymph nodes, kidney, spleen, and hepatic lymph node. Fluorescence microscopy corroborated the ICP-MS results regarding the tissue distribution of QD. The results indicated that (1) ID injected nanoscale QD remained as a deposit in skin and penetrated the surrounding viable subcutis, (2) QD were distributed to draining lymph nodes through the sc lymphatics and to the liver and other organs, and (3) sentinel organs are effective locations for monitoring transdermal penetration of nanoscale materials into animals.

Previously, we reported high hepatotoxic sensitivity of type 2 diabetic (DB) rats to three dissimilar hepatotoxicants. Additional work revealed that a normally nonlethal dose of CCl4 was lethal in DB rats due to inhibited compensatory tissue repair. The present study was conducted to investigate the importance of compensatory tissue repair in determining the final outcome of hepatotoxicity in diabetes, using another structurally and mechanistically dissimilar hepatotoxicant, thioacetamide (TA), to initiate liver injury. A normally nonlethal dose of TA (300 mg/kg, ip), caused 100% mortality in DB rats. Time course studies (0 to 96 h) showed that in the non-DB rats, liver injury initiated by TA as assessed by plasma alanine or aspartate aminotransferase and hepatic necrosis progressed up to 48 h and regressed to normal at 96 h resulting in 100% survival. In the DB rats, liver injury rapidly progressed resulting in progressively deteriorating liver due to rapidly expanding injury, hepatic failure, and 100% mortality between 24 and 48 h post-TA treatment. Covalent binding of 14C-TA-derived radiolabel to liver tissue did not differ from that observed in the non-DB rats, indicating similar bioactivation-based initiation of hepatotoxicity. S-phase DNA synthesis measured by [3H]-thymidine incorporation, and advancement of cells through the cell division cycle measured by PCNA immunohistochemistry, were substantially inhibited in the DB rats compared to the non-DB rats challenged with TA. Thus, inhibited cell division and compromised tissue repair in the DB rats resulted in progressive expansion of liver injury culminating in mortality. In conclusion, it appears that similar to type 1 diabetes, type 2 diabetes also increases sensitivity to dissimilar hepatotoxicants due to inhibited compensatory tissue repair, suggesting that sensitivity to hepatotoxicity in diabetes occurs in the absence as well as presence of insulin.

Streptozotocin (STZ)-induced diabetic (DB) rats are protected from nephrotoxicity of gentamicin, cisplatin and mercuric chloride, although the mechanisms remain unclear. Ninety percent of DB mice receiving a LD90 dose (75 mg/kg, ip) of S-1,2-dichlorovinyl-l-cysteine (DCVC) survived in contrast to only 10% of the nondiabetic (NDB) mice surviving the same dose. We tested the hypothesis that the mechanism of protection is upregulated tissue repair. In the NDB mice, DCVC produced steep temporal increases in blood urea nitrogen (BUN) and plasma creatinine, which were associated with proximal tubular cell (PTC) necrosis, acute renal failure (ARF), and death within 48 h. In contrast, in the DB mice, BUN and creatinine increased less steeply, declining after 36 h to completely resolve by 96 h. HPLC analysis of plasma and urine revealed that DB did not alter the toxicokinetics of DCVC. Furthermore, activity of renal cysteine conjugate β-lyase, the enzyme that bioactivates DCVC, was unaltered in DB mice, undermining the possibility of lower bioactivation of DCVC leading to lower injury. [3H]-thymidine pulse labeling and PCNA analysis indicated an early onset and sustained nephrogenic tissue repair in DCVC-treated DB mice. BRDU immunohistochemistry revealed a fourfold increase in the number of cells in S-phase in the DB kidneys even without exposure to DCVC. Blocking the entry of cells into S-phase by antimitotic intervention using colchicine abolished stimulated nephrogenic tissue repair and nephroprotection. These findings suggest that preplacement of S-phase cells in the kidney due to diabetes is critical in mitigating the progression of DCVC-initiated renal injury by upregulation of tissue repair, leading to survival of the DB mice by avoiding acute renal failure.

Previous studies have shown that renal injury initiated by a lethal dose of S-1,2-dichlorovinyl-l-cysteine (DCVC) progresses due to inhibition of cell division and hence renal repair, leading to acute renal failure (ARF) and death in mice. Renal injury initiated by low to moderate doses of DCVC is repaired by timely and adequate stimulation of renal cell division, tubular repair, restoration of renal structure and function leading to survival of mice. Recent studies have established that mice primed with a low dose of DCVC (15 mg/kg ip) 72 h before administration of a normally lethal dose (75 mg/kg ip) are protected from ARF and death (nephro-autoprotection). We showed that renal cell division and tissue repair stimulated by the low dose are sustained even after the lethal dose administration resulting in survival from ARF and death. If renal cell division induced by the low dose is indeed the critical mechanism of this autoprotection, then its ablation by the antimitotic agent colchicine (1.5 mg CLC/kg ip) should abolish autoprotection. The present interventional experiments were designed to test the hypothesis that DCVC autoprotection is due to stimulated cell division and tissue repair by the priming low dose. CLC intervention at 42 and 66 h after the priming dose resulted in marked progressive elevation of plasma blood urea nitrogen and creatinine resulting in ARF and death of mice. Light microscopic examination of hematoxylin and eosin-stained kidney sections revealed progression of renal necrosis concordant with progressively failing renal function. With CLC intervention, S-phase stimulation (as assessed by BrdU pulse labeling), G1-to-S phase clearance, and cell division were diminished essentially abolishing the promitogenic effect of the priming low dose of DCVC. Phospho-retinoblastoma protein (P-pRB), a crucial protein for S-phase stimulation, and other cellular signaling mechanisms regulating P-pRB were investigated. We report that decreased P-pRB via activation of protein phosphatase-1 by CLC is the critical mechanism of this inhibited S-phase stimulation and ablation of autoprotection with CLC intervention. These findings lend additional support to the notion that stimulated cell division and renal tissue repair by the priming dose of DCVC are the critical mechanisms that allow sustained compensatory tissue repair and survival of mice in nephro-autoprotection.

Vitamin A ( retinol) regulates many biological functions, including epidermal cell growth. Retinyl palmitate ( RP) is the major esterified form of retinol and the predominant component of retinoids in the skin; however, how endogenous levels of RP and retinol in the skin are affected by the age of the animal remains unknown. Furthermore, the levels of retinol and RP in the various skin layers - the stratum corneum, epidermis and dermis of skin - have not been reported. In this paper, we report the development of a convenient method for separation of the skin from SKH-1 female mice into the stratum corneum, epidermis, and dermis and the determination of the levels of RP and retinol in the three fractions by HPLC analysis. The total quantities of RP and retinol from the stratum corneum, epidermis, and dermis are comparable to those extracted from the same amount of intact skin from the same mouse. There was an age-related effect on the levels of RP and retinol in the skin and liver of female mice. An age-related effect was also observed in the stratum corneum, epidermis, and dermis. The levels of RP and retinol were highest in the epidermis of 20-week-old mice, and decreased when the age increased to 60- and 68-weeks. The total amount of RP at 20 weeks of age was found to be 1.52 ng/mg skin, and decreased about 4-fold at 60- and 68-weeks of age. A similar trend was found for the effects of age on the levels of retinol.

Previously, we reported that an ordinarily non-lethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic rats due to inhibited liver tissue repair, whereas 30 mg TA/kg allows 100% survival due to stimulated although delayed tissue repair. Objective of this investigation was to test whether prior administration of a low dose of TA (30 mg/kg) would lead to sustainable stimulation of liver tissue repair in type 1 diabetic rats sufficient to protect from a subsequently administered lethal dose of TA. Therefore, in the present study, the hypothesis that preplacement of tissue repair by a low dose of TA (30 mg TA/kg, ip) can reverse the hepatotoxicant sensitivity (autoprotection) in type 1 diabetic rats was tested. Preliminary studies revealed that a single intraperitoneal (ip) administration of TA causes 90% mortality in diabetic rats with as low as 75 mg/kg. To establish an autoprotection model in diabetic condition, diabetic rats were treated with 30 mg TA/kg (priming dose). Administration of priming dose stimulated tissue repair that peaked at 72 h, at which time these rats were treated with a single ip dose of 75 mg TA/kg. Our results show that tissue repair stimulated by the priming dose enabled diabetic rats to overexpress, calpastatin, endogenous inhibitor of calpain, to inhibit calpain-mediated progression of liver injury induced by the subsequent administration of lethal dose, resulting in 100% survival. Further investigation revealed that protection observed in these rats is not due to decreased bioactivation. These studies underscore the importance of stimulation of tissue repair in the final outcome of liver injury (survival/death) after hepatotoxicant challenge. Furthermore, these results also suggest that it is possible to stimulate tissue repair in diabetics to overcome the enhanced sensitivity of hepatotoxicants.

Previously we have shown that 90% of streptozotocin (STZ)-induced type-1 diabetic (DB) mice survive from acute renal failure (ARF) and death induced by a normally LD(90) dose (75 mg/kg, i.p.) of the nephrotoxicant S-1,2-dichlorovinyl-l-cysteine (DCVC). This remarkable protection is due to a combination of slower progression of DCVC-initiated renal injury and increased compensatory nephrogenic tissue repair in the DB kidneys. BRDU immunohistochemistry revealed that the DB condition led to 4-fold higher number of proximal tubular cells (PTC) entering S-phase of cell cycle. In the present study, we tested the hypothesis that DB-induced augmentation of PTC into S-phase is accompanied by overexpression of the calpain-inhibitor calpastatin, which endogenously prevents the progression of DCVC-initiated renal injury mediated by the calpain escaping out of damaged PTCs. Immunohistochemical detection of renal calpain and its activity in the urine, over a time course after treatment with the LD(90) dose of DCVC, indicated progressive increase in leakage of calpain into the extracellular spaces of the injured PTCs of the non-diabetic (NDB) kidneys as compared to the DB kidneys. Calpastatin expression was minimally detected in the NDB kidneys, using immunohistochemistry, over the time course. On the other hand, consistently higher number of tubules in the DB kidney showed calpastatin expression over the time course. The lower leakage of calpain in the DB kidneys was commensurate with constitutively higher expression of calpastatin in the S-phase-laden PTCs of these mice. To test the protective role of newly divided/dividing PTCs, DB mice were given the anti-mitotic agent colchicine (CLC) (2 mg/kg and 1.5 mg/kg, i.p., on days 8 and 10 after STZ injection) prior to challenge with a LD(90) dose of DCVC, which led to 100% mortality by 48 h. Mortality was due to rapid progression of DCVC-initiated renal injury, suggesting that newly divided/dividing cells are instrumental in mitigating the progression of DCVC-initiated renal injury in DB. The anti-mitotic effect of CLC in DB kidney is associated with lower expression of calpastatin and higher leakage of calpain in the injured tubules. These findings suggest that constitutively higher cell division in the DB kidney is associated with overexpression of calpastatin, which reduces the progression of DCVC-initiated renal injury mediated by calpain on the one hand and accelerates nephrogenic tissue repair on the other, thereby restoring renal structure and function.

Thioacetamide (TA) is bioactivated by CYP2E1 to TA sulfoxide (TASO), and to the highly reactive sulfdioxide (TASO2), which initiates hepatic necrosis by covalent binding. Previously, we have established that TA exhibits saturation toxicokinetics over a 12-fold dose range, which explains the lack of dose–response for bioactivation-based liver injury. In vivo and in vitro studies indicated that the second step (TASO→TASO2) of TA bioactivation is less efficient than the first one (TA→TASO). The objective of the present study was to specifically test the saturation of the second step of TA bioactivation by directly administering TASO, which obviates the contribution from first step, i.e. TA→TASO. Male SD rats were injected with low (50mg/kg, ip), medium (100mg/kg) and high (LD70, 200mg/kg) doses of TASO. Bioactivation-mediated liver injury that occurs in the initial time points (6 and 12h), estimated by plasma ALT, AST and liver histopathology over a time course, was not dose-proportional. Escalation of liver injury thereafter was dose dependent: low dose injury subsided; medium dose injury escalated upto 36h before declining; high dose injury escalated from 24h leading to 70% mortality. TASO was quantified in plasma by HPLC at various time points after administration of the three doses. With increasing dose (i.e., from 50 to 200mg/kg), area under the curve (AUC) and Cmax increased more than dose proportionately, indicating that TASO bioactivation exhibits saturable kinetics. Toxicokinetics and initiation of liver injury of TASO are similar to that of TA, although TASO-initiated injury occurs at lower doses. These findings indicate that bioactivation of TASO to its reactive metabolite is saturable in the rat as suggested by previous studies with TA.

Type 2 diabetic (DB) mice exposed to CCl4 (LD50 = 1.25 ml/kg), acetaminophen (LD80 = 600 mg/kg; APAP), and bromobenzene (LD80 = 0.5 ml/kg) i.p. yielded 30, 20, and 20% mortality, respectively, indicating hepatotoxic resistance. Male Swiss-Webster mice were made diabetic by feeding high fat and administrating streptozotocin (120 mg/kg i.p.) on day 60. On day 71, time-course studies after APAP (600 mg/kg) treatment revealed identical initial liver injury in non-DB and DB mice, which progressed only in non-DB mice, resulting in 80% mortality. The hypothesis that decreased APAP bioactivation, altered toxicokinetics, and/or increased tissue repair are the underlying mechanisms was investigated. High-performance liquid chromatography analysis revealed no difference in plasma and urinary APAP or detoxification of APAP via glucuronidation between DB and non-DB mice. Hepatic CYP2E1 protein and activity, glutathione, and [14C]APAP covalent binding did not differ between DB and non-DB mice, suggesting that lower bioactivation-based injury is not the mechanism of decreased hepatotoxicity in DB mice. Diabetes increased cells in S phase by 8-fold in normally quiescent liver of these mice. Immunohistochemistry revealed overexpression of calpastatin in the newly dividing/divided cells, explaining inhibition of hydrolytic enzyme calpain in perinecrotic areas and lower progression of APAP-initiated injury in the DB mice. Antimitotic intervention of diabetes-associated cell division with colchicine before APAP administration resulted in 70% mortality in APAP-treated colchicine-intervened DB mice. These studies suggest that advancement of cells in the cell division cycle and higher tissue repair protect DB mice by preventing progression of APAP-initiated liver injury that normally leads to mortality.

Tattooing is a popular cosmetic practice involving more than 45 million US citizens. Since the toxicology of tattoo inks and pigments used to formulate tattoo inks has not been reported, we studied the immunological impact of tattooing and determined recovery time from this trauma. SKH-1 hairless mice were tattooed using commercial tattoo inks or suspensions of titanium dioxide, cadmium sulfide, or iron oxide, and sacrificed at 0.5, 1, 3, 4, 7, or 14 days post-tattooing. Histological evaluation revealed dermal hemorrhage at 0.5 and 1 day. Acute inflammation and epidermal necrosis were initiated at 0.5 day decreasing in incidence by day 14. Dermal necrosis and epidermal hyperplasia were prominent by day 3, reducing in severity by day 14. Chronic active inflammation persisted in all tattooed mice from day 3 to 14 post-tattooing. Inguinal and axillary lymph nodes were pigmented, the inguinal being most reactive as evidenced by lymphoid hyperplasia and polymorphonuclear infiltration. Cutaneous nuclear protein concentrations of nuclear factor-kappa B were elevated between 0.5 and 4 days. Inflammatory and proliferative biomarkers, cyclooxygenase-1, cyclooxygenase-2, and ornithine decarboxylase protein levels were elevated between 0.5 and 4 days in the skin and decreased to control levels by day 14. Interleukin-1 beta and interleukin-10 were elevated in the lymph nodes but suppressed in the tattooed skin, with maximal suppression occurring between days 0.5 and 4. These data demonstrate that mice substantially recover from the tattooing insult by 14 days, leaving behind pigment in the dermis and the regional lymph nodes. The response seen in mice is similar to acute injury seen in humans, suggesting that the murine model might be a suitable surrogate for investigating the toxicological and phototoxicological properties of ingredients used in tattooing.

We have investigated the effects of chlordecone 1(CD)+CCl4 combination in adult (3 months), middle aged (14 months), and old aged (24 months) male Fischer 344 (F344) rats. After a non-toxic dietary regimen of CD (10 ppm) or normal powdered diet for 15 days, rats received a single non-toxic dose of CCl4 (100 microl/kg, i.p., 1:4 in corn oil) or corn oil (500 microl/kg, i.p.) alone on day 16. Liver injury was assessed by plasma ALT, AST, and histopathology during a time course of 0-96 h. Liver tissue repair was measured by [3H-CH3]-thymidine (3H-T) incorporation into hepatic nuclear DNA and proliferating cell nuclear antigen (PCNA) immunohistochemistry. Hepatomicrosomal CYP2E1 protein, enzyme activity, and covalent binding of 14CCl4-derived radiolabel were measured in normal and CD fed rats. Exposure to CCl4 alone caused modest liver injury only in 14- and 24-month-old rats but neither progression of injury nor mortality. The CD+CCl4 combination led to 100% mortality in 3-month-old rats by 72 h, whereas none of the 14- and 24-month-old rats died. Both 3- and 14-month-old rats exposed to CD+Cl4 had identical liver injury up to 36 h indicating that bioactivation-mediated CCl4 injury was the same in the two age groups. Thereafter, liver injury escalated only in 3-month-old while it declined in 14-month-old rats. In 24-month-old rats initial liver injury at 6 h was similar to the 3- and 14-month-old rats and thereafter did not develop to the level of the other two age groups, recovering from injury by 96 h as in the 14-month-old rats. Neither hepatomicrosomal CYP2E1 protein nor the associated p-nitrophenol hydroxylase activity or covalent binding of 14CCl4-derived radiolabel to liver tissue differed between the age groups or diet regimens 2 h after the administration of 14CCl4. Compensatory liver tissue repair (3H-T, PCNA) was prompt and robust soon after CCl4 liver injury in the 14- and 24-month-old rats. In stark contrast, in the 3-month-old rats it failed allowing unabated progression of liver injury. These findings suggest that stimulation of early onset and robust liver tissue repair rescue the 14- and 24-month-old F344 rats from the lethal effect of the CD+CCl4 combination.