Gary J. Overmann

Identification and Characterization of Toxicity of Contaminants in Pet Food Leading to an Outbreak of Renal Toxicity in Cats and Dogs

This paper describes research relating to the major recall of pet food that occurred in Spring 2007 in North America. Clinical observations of acute renal failure in cats and dogs were associated with consumption of wet pet food produced by a contract manufacturer producing for a large number of companies. The affected lots of food had been formulated with wheat gluten originating from China. Pet food and gluten were analyzed for contaminants using several configurations of high-performance liquid chromatography (HPLC) and mass spectrometry (MS), which revealed a number of simple triazine compounds, principally melamine and cyanuric acid, with lower concentrations of ammeline, ammelide, ureidomelamine, and N-methylmelamine. Melamine and cyanuric acid, have been tested and do not produce acute renal toxicity. Some of the triazines have poor solubility, as does the compound melamine cyanurate. Pathological evaluation of cats and dogs that had died from the acute renal failure indicated the presence of crystals in kidney tubules. We hypothesized that these crystals were composed of the poorly soluble triazines, a melamine-cyanuric acid complex, or a combination. Sprague dawley rats were given up to 100 mg/kg ammeline or ammelide alone, a mixture of melamine and cyanuric acid (400/400 mg/kg/day), or a mixture of all four compounds (400 mg/kg/day melamine, 40 mg/kg/day of the others). Neither ammeline nor ammelide alone produced any renal effects, but the mixtures produced significant renal damage and crystals in nephrons. HPLC-MS/MS confirmed the presence of melamine and cyanuric acid in the kidney. Infrared microspectroscopy on individual crystals from rat or cat (donated material from a veterinary clinic) kidneys confirmed that they were melamine-cyanuric acid cocrystals. Crystals from contaminated gluten produced comparable spectra. These results establish the causal link between the contaminated gluten and the adverse effects and provide a mechanistic explanation for how two apparently innocuous compounds could have adverse effects in combination, that is, by forming an insoluble precipitate in renal tubules leading to progressive tubular blockage and degeneration.

The role of metallothionein induction and altered zinc status in maternally mediated developmental toxicity: Comparison of the effects of urethane and styrene in rats

We hypothesize that maternal metallothionein (MT) induction by toxic dosages of chemicals may contribute to or cause developmental toxicity by a chain of events leading to a transient but developmentally adverse decrease in Zn availability to the embryo. This hypothesis was tested by evaluating hepatic MT induction, maternal and embryonic Zn status, and developmental toxicity after exposure to urethane, a developmental toxicant, or styrene, which is not a developmental toxicant. Pregnant Sprague-Dawley rats were given 0 or 1 g/kg urethane ip, or 0 or 300 mg/kg styrene in corn oil po, on Gestation Day 11 (sperm positive = Gestation Day 0). These were maternally toxic dosages. As both treatments decreased food consumption, separate pair-fed control groups were also evaluated for effects on MT and Zn status and development. In addition, Gestation Day 11 rat embryos were exposed to urethane in vitro in order to determine whether urethane has the potential to be directly embryotoxic. Urethane treatment induced hepatic MT 14-fold over control; styrene treatment induced MT 2.5-fold. The MT induction by styrene could be attributed to decreased food intake, as a similar level of induction was observed in a pair-fed untreated control group. However, the level of MT induction by urethane was much greater than that produced by decreased food intake alone. Hepatic Zn concentration, particularly in the cytosol, was increased in the presence of increased hepatic MT concentration. Plasma Zn concentration was significantly decreased (approximately 30%) by urethane treatment, but not by styrene or food restriction (pair-feeding). Distribution of 65Zn to the liver of urethane-treated dams was significantly greater (by 30%), while distribution to embryonic tissues was significantly lower (by at least 50%) than in pair-fed or ad lib.-fed controls. Styrene treatment had no effect on 65Zn distribution. Urethane was developmentally toxic, causing an 18% decrease in fetal weight and a significant delay in skeletal ossification, but was not toxic to rat embryos in vitro. Styrene was not developmentally toxic. The changes observed after urethane treatment, namely substantial hepatic MT induction and altered maternal and embryonic Zn status, along with the lack of direct embryotoxicity of urethane in vitro, support the hypothesis that these maternal effects contribute to developmental toxicity. The lack of similar changes in styrene-intoxicated dams provides one explanation for its low developmental toxicity at maternally toxic dosages.

Altered Zn status by α-hederin in the pregnant rat and its relationship to adverse developmental outcome

The hypothesis that an acute-phase reaction in the pregnant animal causes a systemic redistribution of Zn, resulting in a transient but developmentally adverse Zn deficiency in the embryo, was tested by treating pregnant rats during organogenesis with alpha-hederin, an agent reported to induce substantial metallothionein (MT) synthesis in rat liver, and determining hepatic MT concentration, hepatic and plasma Zn concentration, and systemic distribution of a pulse of 65Zn after treatment. Developmental toxicity was assessed by evaluating morphologic development in term fetuses. A single dose of alpha-hederin, injected sc at dosages of 3 to 300 mumol/kg, caused an acute phase response, indicated by decreased Fe and Zn, and increased Cu, alpha 1-acid glycoprotein, and ceruloplasmin concentration in plasma, along with a dosage-related increase in maternal hepatic MT concentration. The maximum induction of MT was 11 to 15-fold greater than control and occurred at dosages of 30 mumol/kg and higher, and MT concentration reached its peak 12 to 24 h after treatment. Zn concentration in liver and liver cytosol increased along with MT, reaching a maximum level at dosages of 30 mumol/kg and higher. Plasma Zn concentration decreased after alpha-hederin treatment to a level approximately 75% of control at a dosage of 30 mumol/kg and 50% of control at 300 mumol/kg. Therefore, hepatic MT induction was associated with most, but not all, of the decrease in plasma Zn concentration. Zn distribution was evaluated by giving an oral pulse of 65Zn 8 h after treatment with 0, 30, or 300 mumol/kg alpha-hederin on gestation day 11, and measuring 65Zn levels 18 h after treatment. The fraction of 65Zn distributed to the liver of treated rats (either dosage) was twice that of control, but distribution of 65Zn to other maternal tissues was decreased. 65Zn accumulation by conceptuses was significantly decreased, attributable to decreased accumulation in decidua, but not in visceral yolk sacs or embryos; however, at this stage of development the decidua accounts for a greater quantity of Zn than either of the other products of conception and may serve as the Zn-storing tissue for the conceptus. Both 30 and 300 mumol/kg increased resorption incidence, and 300 mumol/kg also decreased fetal weight and increased the incidence of abnormal fetuses. Serum collected from rats two hours after alpha-hederin treatment (i.e., before the onset of MT synthesis) supported rat embryo development in vitro, whereas serum collected 18 h after treatment did not. Adding Zn to this serum restored normal embryonic development.(ABSTRACT TRUNCATED AT 400 WORDS)

Impact of the phytoestrogen content of laboratory animal feed on the gene expression profile of the reproductive system in the immature female rat

The effect of the dietary background of phytoestrogens on the outcome of rodent bioassays used to identify and assess the reproductive hazard of endocrine-disrupting chemicals is controversial. Phytoestrogens, including genistein, daidzein, and coumestrol, are fairly abundant in soybeans and alfalfa, common ingredients of laboratory animal diets. These compounds are weak agonists for the estrogen receptor (ER) and, when administered at sufficient doses, elicit an estrogenic response in vivo. In this study, we assessed the potential estrogenic effects of dietary phytoestrogens at the gene expression level, together with traditional biologic end points, using estrogen-responsive tissues of the immature female rat. We compared the gene expression profile of the uterus and ovaries, as a pool, obtained using a uterotrophic assay protocol, from intact prepubertal rats fed a casein-based diet (free from soy and alfalfa) or a regular rodent diet (Purina 5001) containing soy and alfalfa. Estrogenic potency of the phytoestrogen-containing diet was determined by analyzing uterine wet weight gain, luminal epithelial cell height, and gene expression profile in the uterus and ovaries. These were compared with the same parameters evaluated in animals exposed to a low dose of a potent ER agonist [0.1 μg/kg/day 17α-ethynyl estradiol (EE) for 4 days]. Exposure to dietary phytoestrogens or to a low dose of EE did not advance vaginal opening, increase uterine wet weight, or increase luminal epithelial cell height in animals fed either diet. Although there are genes whose expression differs in animals fed the soy/alfalfa-based diet versus the casein diet, those genes are not associated with estrogenic stimulation. The expression of genes well known to be estrogen regulated, such as progesterone receptor, intestinal calcium-binding protein, and complement component 3, is not affected by consumption of the soy/alfalfa-based diet when assessed by microarray or quantitative reverse transcriptase–polymerase chain reaction analysis. Our results indicate that although diet composition has an impact on gene expression in uterus and ovaries, it does not contribute to the effects of an ER agonist.

Effects of Transplacental 17-α-Ethynyl Estradiol or Bisphenol A on the Developmental Profile of Steroidogenic Acute Regulatory Protein in the Rat Testis

Previous research from our laboratory has determined the transcript profiles for developing fetal rat female and male reproductive tracts following transplacental exposure to estrogens. Prenatal exposure to bisphenol A (BPA) or 17-α-ethynyl estradiol (EE) significantly affects steroidogenic acute regulatory (StAR) protein transcript levels in the developing male rat reproductive tract. The purpose of this study was to establish the intratesticular distribution and temporal expression pattern of StAR, a key gene involved in steroidogenesis. Beginning on gestation day (GD) 11, pregnant Sprague-Dawley rats were exposed daily to 10μg/kg/day EE and fetal testes were harvested at GD16, 18, or 20. Quantitative reverse transcriptase PCR (QRT-PCR) demonstrated no significant difference in StAR transcript levels present at GD16. However, at GD18, StAR transcripts were significantly decreased following exposure. Immunohistochemistry demonstrated similar StAR protein levels in interstitial region of GD16 testes and an obvious decrease in StAR protein levels in the interstitial region of GD18 testes. Moreover, starting at GD11 additional dams were dosed with 0.001 or 0.1 μg/kg/day EE or 0.02, 0.5, 400 mg/kg/day BPA via subcutaneous injections. QRT-PCR validated previous microarray dose-related decreases in StAR transcripts at GD20, whereas immunohistochemistry results demonstrated decreases in StAR protein levels in the interstitial region at the highest EE and BPA doses only. Neither EE nor BPA exposure caused morphological changes in the developing seminiferous cords, Sertoli cells, gonocytes, or the interstitial region or Leydig cells at GD16-20. High levels of estrogens decrease StAR expression in the fetal rat testis during late gestation.

Repeated administration of α-hederin results in alterations in maternal zinc status and adverse developmental outcome in the rat

The administration of alpha-hederin, an inducer of metallothionein, results in a secondary zinc deficiency that may be an important maternally mediated mechanism of developmental toxicity. Previous studies have shown adverse developmental outcome with a single administration of alpha-hederin to rats on gestation day (GD) 8 or 11. The objective of this study was to determine whether dosing of alpha-hederin throughout organogenesis would result in a sustained elevation of maternal hepatic metallothionein and subsequent developmental abnormalities. Rats were administered dosage levels of 0 (vehicle only), 20, or 30 mumol/kg from GD 6-15. Maternal hepatic metallothionein levels were 10-fold higher on GD 16 in the treatment groups than the controls. Consequently, liver zinc concentrations increased 60% and 54%, whereas plasma levels decreased 23% and 33% in the 20 and 30 mumol/kg treatment groups, respectively. At GD 20, mean fetal weights of the treatment litters were 11% less than control litters. The administration of alpha-hederin resulted in a threefold increase in the number of offspring that exhibited developmental abnormalities, including visceral and skeletal malformations. Following an oral pulse of 65Zn subsequent to treatment with 0 or 20 mumol/kg of alpha-hederin, the distribution of 65Zn to the liver of treated dams was twice that of controls, whereas the radiolabeled zinc apportioned to the decidua and uterus decreased by 44%. Furthermore, the 65Zn detected in the embryos from treated dams was 70% lower than in embryos from control dams. In conclusion, low doses of a metallothionein inducer administered to the dam from GD 6-15 resulted in a sustained elevation of hepatic metallothionein and a subsequent redistribution of zinc leading to a decrease in the zinc available to the embryo and ultimately to adverse development of the offspring. Repeated dosing throughout organogenesis, as required in regulated safety assessment testing, increased the severity of the effects previously observed with single large dosages of the toxicant administered during midgestation.

Grouping 34 Chemicals Based on Mode of Action Using Connectivity Mapping

Connectivity mapping is a method used in the pharmaceutical industry to find connections between small molecules, disease states, and genes. The concept can be applied to a predictive toxicology paradigm to find connections between chemicals, adverse events, and genes. In order to assess the applicability of the technique for predictive toxicology purposes, we performed gene array experiments on 34 different chemicals: bisphenol A, genistein, ethinyl-estradiol, tamoxifen, clofibrate, dehydorepiandrosterone, troglitazone, diethylhexyl phthalate, flutamide, trenbolone, phenobarbital, retinoic acid, thyroxine, 1α,25-dihydroxyvitamin D3, clobetasol, farnesol, chenodeoxycholic acid, progesterone, RU486, ketoconazole, valproic acid, desferrioxamine, amoxicillin, 6-aminonicotinamide, metformin, phenformin, methotrexate, vinblastine, ANIT (1-naphthyl isothiocyanate), griseofulvin, nicotine, imidacloprid, vorinostat, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) at the 6-, 24-, and 48-hour time points for 3 different concentrations in the 4 cell lines: MCF7, Ishikawa, HepaRG, and HepG2 GEO (super series accession no.: GSE69851). The 34 chemicals were grouped in to predefined mode of action (MOA)-based chemical classes based on current literature. Connectivity mapping was used to find linkages between each chemical and between chemical classes. Cell line-specific linkages were compared with each other and to test whether the method was platform and user independent, a similar analysis was performed against publicly available data. The study showed that the method can group chemicals based on MOAs and the inter-chemical class comparison alluded to connections between MOAs that were not predefined. Comparison to the publicly available data showed that the method is user and platform independent. The results provide an example of an alternate data analysis process for high-content data, beneficial for predictive toxicology, especially when grouping chemicals for read across purposes.

A novel transcriptomics based in vitro method to compare and predict hepatotoxicity based on mode of action.

High-content data have the potential to inform mechanism of action for toxicants. However, most data to support this notion have been generated in vivo. Because many cell lines and primary cells maintain a differentiated cell phenotype, it is possible that cells grown in culture may also be useful in predictive toxicology via high-content approaches such as whole-genome microarray. We evaluated global changes in gene expression in primary rat hepatocytes exposed to two concentrations of ten hepatotoxicants: acetaminophen (APAP), β-naphthoflavone (BNF), chlorpromazine (CPZ), clofibrate (CLO), bis(2-ethylhexyl)phthalate (DEHP), diisononyl phthalate (DINP), methapyrilene (MP), valproic acid (VPA), phenobarbital (PB) and WY14643 at two separate time points. These compounds were selected to cover a range of mechanisms of toxicity, with some overlap in expected mechanism to address the question of how predictive gene expression analysis is, for a given mode of action. Gene expression microarray analysis was performed on cells after 24h and 48h of exposure to each chemical using Affymetrix microarrays. Cluster analysis suggests that the primary hepatocyte model was capable of responding to these hepatotoxicants, with changes in gene expression that appear to be mode of action-specific. Among the different methods used for analysis of the data, a combination method that used pathways (MOAs) to filter total probesets provided the most robust analysis. The analysis resulted in the phthalates clustering closely together, with the two other peroxisome proliferators, CLO and WY14643, eliciting similar responses at the whole-genome and pathway levels. The Cyp inducers PB, MP, CPZ and BNF also clustered together. VPA and APAP had profiles that were unique. A similar analysis was performed on externally available (TG-GATES) in vivo data for 6 of the chemicals (APAP, CLO, CPZ, MP, MP and WY14643) and compared to the in vitro result. These results indicate that transcription profiling using an in vitro assay may offer pertinent biological data to support predictions of in vivo hepatotoxicity potential.