Alan Warbritton

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.

Estrogen-Induced Rat Breast Carcinogenesis is Characterized by Alterations in DNA Methylation, Histone Modifications, and Aberrant microRNA Expression

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.

Dose-response assessment of nephrotoxicity from a 7-day combined exposure to melamine and cyanuric acid in F344 rats.

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.

The role of programmed cell death in the toxicity of the mutagens, ethyl methanesulfonate and N-ethyl-N'-nitrosourea, in AHH-1 human lymphoblastoid cells

In order to determine the pathway for cell death in alkylating agent-exposed human lymphoblastoid cells, AHH-1 cells were exposed to either ethyl methanesulfonate (EMS) or ethyl nitrosourea (ENU) and the effect on relative cell growth and plating efficiency quantified. Flow cytometric (FCM) assays were utilized to quantify cell viability and to determine if cell death occurred through necrosis or apoptosis. As expected, exposure to the simple ethylating agents resulted in concentration-dependent decreases in plating efficiencies at each time interval after exposure (Days 0, 2, 3 and 7). EMS exposure did not significantly affect the relative cell growth, in contrast to ENU exposure, which inhibited cell growth. The FCM viability assay, based on light scatter characteristics, revealed that exposure to either alkylating agent resulted in a significant reduction in the percentage of viable cells. The results of the FCM dye-exclusion assays revealed that while necrosis occurred in EMS- and ENU-exposed cells, the primary manner of cell death was apoptosis. AHH-1 cells were stained with propidium iodide and fluorescein diacetate, the population of cells sorted electronically and the cell type (necrotic, apoptotic or viable) confirmed morphologically. Our results clearly indicate that exposure to EMS or ENU results in the movement of AHH-1 cells into the pathway for apoptosis and cell death.

The mRNA expression and histological integrity in rat forebrain motor and sensory regions are minimally affected by acrylamide exposure through drinking water.

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.

Age-Related Susceptibility to Chlordecone-Potentiated Carbon Tetrachloride Hepatotoxicity and Lethality Is Due to Hepatic Quiescence

ABSTRACT: Previous studies revealed that postnatally developing rats are resilient to the lethal effects of chlordecone (CD) + carbon letrachloride (CCI4) combination. The objective of this study was to investigate the underlying mechanism. We hypothesized that ongoing cell division and cell cycle progression as well as additional toxicant-induced stimulation of tissue repair help in restraining the progression of injury on the one hand, and in recovery through speedy healing on the other. Postnatally developing (20− and 45-d) and adult (60-d) male Sprague-Dawley rats were challenged with a nontoxic single dose of CCI4 (100 μL/kg. i.p.) or corn oil after pretreatment with either dietary CD (10 ppm) or normal diet (ND) for 15 d. Hepatocellular injury was assessed by measuring serum enzymes [alanine transaminase (ALT), sorbitol dehydrogenase (SDH)], and bilirubin, as well as by histopathologic examination of liver sections during a time course of 0–96 h after the administration of CCI4 or corn oil. Hepatocellular regeneration was assessed by [3H]thymidine ([3H]T) incorporation into hepatic nuclear DNA. In CD + CCI4 treatment, ALT, SDH, and bilirubin levels peaked between 36 and 48 h after CCI4. All 20-d-old rats survived the challenge of CD + CCI4. CD-potentiated hepatotoxicity and lethality of CCI4 begin to be manifested in 45-d-old rats at 48 h and later times (25% mortality), whereas adult rats experience progressive hepatotoxic injury and 100% mortality by 72 h. In contrast, regardless of pretreatment, 20-d-old rats recover fully from injury by 72 h after CCI4 treatment. The rapid recovery of 20-d-old rats was associated with a combination of higher level of ongoing cell division and additional sustained stimulation of [3H]T incorporation from 24 to 72 h after the administration of CCI4. In the older rats (45− or 60-d-old) this response was significantly delayed and attenuated. Ongoing cell division and CCI4-stimulated regeneration were inversely related to postnatal development (20-, 45-, or 60-d). These biochemical, histopathological, and [3H]T incorporation studies suggest that the liver of younger rats has greater plasticity for repair after toxic injury whereas adult liver is much more quiescent in this regard.

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.

Levels of retinyl palmitate and retinol in stratum corneum, epidermis and dermis of SKH-1 mice.

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.

Populations of p53 codon 270 CGT to TGT mutant cells in SKH-1 mouse skin tumors induced by simulated solar light.

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 ∼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 × 10−3 to 9.42 × 10−2, with 3.26 × 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.

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.