Lora L. Arnold

Methylated Arsenicals: The Implications of Metabolism and Carcinogenicity Studies in Rodents to Human Risk Assessment

Monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) are active ingredients in pesticidal products used mainly for weed control. MMAV and DMAV are also metabolites of inorganic arsenic, formed intracellularly, primarily in liver cells in a metabolic process of repeated reductions and oxidative methylations. Inorganic arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder, and lung. However, a good animal model has not yet been found. Although the metabolic process of inorganic arsenic appears to enhance the excretion of arsenic from the body, it also involves formation of methylated compounds of trivalent arsenic as intermediates. Trivalent arsenicals (whether inorganic or organic) are highly reactive compounds that can cause cytotoxicity and indirect genotoxicity in vitro. DMAV was found to be a bladder carcinogen only in rats and only when administered in the diet or drinking water at high doses. It was negative in a two-year bioassay in mice. MMAV was negative in 2-year bioassays in rats and mice. The mode of action for DMAV-induced bladder cancer in rats appears to not involve DNA reactivity, but rather involves cytotoxicity with consequent regenerative proliferation, ultimately leading to the formation of carcinoma. This critical review responds to the question of whether DMAV-induced bladder cancer in rats can be extrapolated to humans, based on detailed comparisons between inorganic and organic arsenicals, including their metabolism and disposition in various animal species. The further metabolism and disposition of MMAV and DMAV formed endogenously during the metabolism of inorganic arsenic is different from the metabolism and disposition of MMAV and DMAV from exogenous exposure. The trivalent arsenicals that are cytotoxic and indirectly genotoxic in vitro are hardly formed in an organism exposed to MMAV or DMAV because of poor cellular uptake and limited metabolism of the ingested compounds. Furthermore, the evidence strongly supports a nonlinear dose-response relationship for the biologic processes involved in the carcinogenicity of arsenicals. Based on an overall review of the evidence, using a margin-of-exposure approach for MMAV and DMAV risk assessment is appropriate. At anticipated environmental exposures to MMAV and DMAV, there is not likely to be a carcinogenic risk to humans.

Evaluation of the carcinogenicity of inorganic arsenic

Abstract Inorganic arsenic (iAs) at high exposures is a human carcinogen, affecting mainly the urinary bladder, lung and skin. We present an assessment of the mode of action (MOA) of iAs’s carcinogenicity based on the United States Environmental Protection Agency/International Programme on Chemical Safety (USEPA/IPCS) framework, focusing primarily on bladder cancer. Evidence is presented for a MOA involving formation of reactive trivalent metabolites interacting with critical cellular sulfhydryl groups, leading to cytotoxicity and regenerative cell proliferation. Metabolism, kinetics, cell transport, and reaction with specific proteins play a critical role in producing the effects at the cellular level, regardless of cell type, whether urothelium, lung epithelium or epidermis. The cytotoxicity induced by iAs results in non-cancer toxicities, and the regenerative cell proliferation enhances development of epithelial cancers. In other tissues, such as vascular endothelium, different toxicities develop, not cancer. Evidence supporting this MOA comes from in vitro investigations on animal and human cells, from animal models, and from epidemiological studies. This MOA implies a non-linear, threshold dose-response relationship for both non-cancer and cancer end points. The no effect levels in animal models (approximately 1 ppm of water or diet) and in vitro (>0.1 µM trivalent arsenicals) are strikingly consistent. Cancer effects of iAs in humans generally are not observed below exposures of 100–150 ppb in drinking water: below these exposures, human urine concentrations of trivalent metabolites are generally below 0.1 µM, a concentration not associated with bladder cell cytotoxicity in in vitro or animal models. Environmental exposures to iAs in most of the United States do not approach this threshold.

Binding of dimethylarsinous acid to Cys-13α of rat hemoglobin is responsible for the retention of arsenic in rat blood

The metabolism, disposition, and carcinogenicity of arsenic differ dramatically between humans and rats. To understand the molecular basis of these differences, we have characterized arsenic species in rats that were treated with inorganic arsenate (iAsV), monomethylarsonic acid (MMAV), or dimethylarsinic acid (DMAV) for up to 15 weeks. Arsenic significantly accumulated in the red blood cells (RBCs) of rats in the form of hemoglobin (Hb) complexed with dimethylarsinous acid (DMAIII), regardless of whether the rats were treated with iAsV, MMAV, or DMAV, suggesting rapid methylation of arsenic species followed by strong binding of DMAIII to rat Hb. The binding site for DMAIII was identified to be cysteine 13 in the alpha-chain of rat Hb with a stoichiometry of 1:1. Over 99% of the total arsenic (maximum 2.5-3.5 mM) in rat RBCs was bound to Hb for all rats examined (n = 138). In contrast, only 40-49% of the total arsenic (maximum approximately 10 muM) in rat plasma was bound to proteins. The ratios of the total arsenic in RBCs to that in plasma ranged from 88-423 for rats that were fed iAsV, 100-680 for rats that were fed MMAV, and 185-1393 for rats that were fed DMAV, when samples were obtained over the 15-week exposure duration. Previous studies have shown an increase in urothelial hyperplasia in rats fed DMAV. This is the first article reporting that treatment with iAsV in the drinking water also produces urothelial hyperplasia and at an even earlier time point than dietary DMAV. Dietary MMAV produced only a slight urothelial response. A correlation between the Hb-DMAIII complex and urothelial lesion severity in rats was observed. The lack of cysteine 13alpha in human Hb may be responsible for the shorter retention of arsenic in human blood. These differences in the disposition of arsenicals may contribute to the observed differences between humans and rats in susceptibility to arsenic carcinogenicity.

Effects of Pioglitazone, a Peroxisome Proliferator-Activated Receptor Gamma Agonist, on the Urine and Urothelium of the Rat

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, which belong to the nuclear receptor superfamily. Some PPARgamma agonists, such as pioglitazone, and dual PPARgamma/PPARalpha agonists, such as muraglitazar, induced urothelial bladder tumors in rats but not in mice. In this study, we investigated the early effects in the urine and bladder of rats treated with pioglitazone to evaluate the possible relation between urinary solids formation and urothelial cytotoxicity and regenerative proliferation. In a 4-week experiment, treatment of rats with 16 mg/kg pioglitazone induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation measured by bromodeoxyuridine labeling index and hyperplasia by histology. It also produced alterations in urinary solid formation, especially calcium-containing crystals and calculi. PPARgamma agonists (pioglitazone and troglitazone) in vitro reduced rat urothelial cell proliferation and induced uroplakin synthesis, a specific differentiation marker in urothelial cells. Our data support the hypothesis that the bladder tumors produced in rats by pioglitazone are related to the formation of urinary solids. This strongly supports the previous conclusion in studies with muraglitazar that this is a rat-specific phenomenon and does not pose a urinary bladder cancer risk to humans treated with these agents.

Investigations of Rodent Urinary Bladder Carcinogens: Collection, Processing, and Evaluation of Urine and Bladders:

Examination of the urine and the bladder epithelium are essential to the investigation of mechanisms of urinary bladder carcinogens in rodents. However, urine and bladder tissue specimens must be collected and processed properly if accurate data are to be derived. The optimum specimen for analysis of urinary constituents is fresh void urine collected from nonfasting animals. Fasting the animal prior to urine collection changes the normal composition, including pH. Many of the normal urinary constituents can influence the mode of action of bladder carcinogens, especially for non-genotoxic agents. Light microscopy is routinely used to examine the bladder epithelium. However, it is often necessary to use more sensitive techniques, such as scanning electron microscopy (SEM) to detect subtle cytotoxic changes in the superficial cell layer of the urothelium, and bromodeoxyuridine (BrdU) incorporation, PCNA, or Ki-67 immunohistochemistry to determine the labeling index for cell proliferation. The urinary bladder must be handled gently and inflated with fixative in situ before the animal dies to avoid artifactual autolytic damage to the bladder epithelium that is visible by SEM and may be mistaken for treatment-related changes. The purpose of this paper is to provide information for the proper collection and examination of urine and the urinary bladder.

Dissimilatory arsenate reductase activity and arsenate-respiring bacteria in bovine rumen fluid, hamster feces, and the termite hindgut

Abstract Bovine rumen fluid and slurried hamster feces completely reduced millimolar levels of arsenate to arsenite upon incubation under anoxic conditions. This activity was strongly inhibited by autoclaving or aerobic conditions, and partially inhibited by tungstate or chloramphenicol. The rate of arsenate reduction was faster in feces from a population of arsenate-watered (100 ppm) hamsters compared to a control group watered without arsenate. Using radioisotope methods, arsenate reductase activity in hamster feces was also detected at very low concentrations of added arsenate ( approximately 10 muM). Bacterial cultures were isolated from these materials, as well as from the termite hindgut, that grew using H(2) as their electron donor, acetate as their carbon source, and arsenate as their respiratory electron acceptor. The three cultures aligned phylogenetically either with well-established enteric bacteria, or with an organism associated with feedlot fecal wastes. Because arsenite is transported across the gut epithelium more readily than arsenate, microbial dissimilatory reduction of arsenate in the gut may promote the bodys absorption of arsenic and hence potentiate its toxicity.

Dietary administration of sodium arsenite to rats: Relations between dose and urinary concentrations of methylated and thio-metabolites and effects on the rat urinary bladder epithelium

Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in drinking water is carcinogenic to humans, inducing skin, urinary bladder and lung tumors. In vivo, inorganic arsenic is metabolized to organic methylated arsenicals including the highly toxic dimethylarsinous acid (DMA(III)) and monomethylarsonous acid (MMA(III)). Short-term treatment of rats with 100 microg/g trivalent arsenic (As(III)) as sodium arsenite in the diet or in drinking water induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation and hyperplasia. The objectives of this study were to determine if these arsenic-induced urothelial effects are dose responsive, the dose of arsenic at which urothelial effects are not detected, and the urinary concentrations of the arsenical metabolites. We treated female F344 rats for 5 weeks with sodium arsenite at dietary doses of 0, 1, 10, 25, 50, and 100 ppm. Cytotoxicity, cell proliferation and hyperplasia of urothelial superficial cells were increased in a dose-responsive manner, with maximum effects found at 50 ppm As(III). There were no effects at 1 ppm As(III). The main urinary arsenical in As(III)-treated rats was the organic arsenical dimethylarsinic acid (DMA(V)). The thio-metabolites dimethylmonothioarsinic acid (DMMTA(V)) and monomethylmonothioarsinic acid (MMMTA(V)) were also found in the urine of As(III)-treated rats. The LC(50) concentrations of DMMTA(V) for rat and human urothelial cells in vitro were similar to trivalent oxygen-containing arsenicals. These data suggest that dietary As(III)-induced urothelial cytotoxicity and proliferation are dose responsive, and the urothelial effects have a threshold corresponding to the urinary excretion of measurable reactive metabolites.

Chronic studies evaluating the carcinogenicity of monomethylarsonic acid in rats and mice

Monomethylarsonic acid (MMA) was administered in the diet of male and female Fischer F344 rats and B6C3F1 mice in 2-year feeding studies according to US EPA guidelines. Rats were treated with 50, 400, or 1300 ppm MMA and mice were treated with 10, 50, 200, or 400 ppm MMA based on preliminary short-term studies. The highest dose in the male and female rat groups was reduced to 1000 ppm during week 53 and then further reduced to 800 ppm during week 60 due to high mortality in the male rats. There was no treatment-related mortality in the mice. The primary target organ for MMA-induced toxicity in rats and mice was the large intestine. Toxicity was more severe in rats compared to mice and in male rats compared to female rats. The maximum tolerated dose for chronic dietary administration of MMA in rats and mice was assessed as 400 ppm, and the no effect level with regard to intestinal toxicity was assessed as 50 ppm for rats and female mice and 200 ppm for male mice. There were no treatment-related neoplastic effects detected in either the rat or the mouse.

Mouse arsenic (+3 oxidation state) methyltransferase genotype affects metabolism and tissue dosimetry of arsenicals after arsenite administration in drinking water

Arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes methylation of inorganic arsenic (iAs) producing a number of methylated arsenic metabolites. Although methylation has been commonly considered a pathway for detoxification of arsenic, some highly reactive methylated arsenicals may contribute to toxicity associated with exposure to inorganic arsenic. Here, adult female wild-type (WT) C57BL/6 mice and female As3mt knockout (KO) mice received drinking water that contained 1, 10, or 25 ppm (mg/l) of arsenite for 33 days and blood, liver, kidney, and lung were taken for arsenic speciation. Genotype markedly affected concentrations of arsenicals in tissues. Summed concentrations of arsenicals in plasma were higher in WT than in KO mice; in red blood cells, summed concentrations of arsenicals were higher in KO than in WT mice. In liver, kidney, and lung, summed concentrations of arsenicals were greater in KO than in WT mice. Although capacity for arsenic methylation is much reduced in KO mice, some mono-, di-, and tri-methylated arsenicals were found in tissues of KO mice, likely reflecting the activity of other tissue methyltransferases or preabsorptive metabolism by the microbiota of the gastrointestinal tract. These results show that the genotype for arsenic methylation determines the phenotypes of arsenic retention and distribution and affects the dose- and organ-dependent toxicity associated with exposure to inorganic arsenic.

Severe systemic toxicity and urinary bladder cytotoxicity and regenerative hyperplasia induced by arsenite in arsenic (+3 oxidation state) methyltransferase knockout mice. A preliminary report.

Arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes reactions which convert inorganic arsenic to methylated metabolites. This study determined whether the As3mt null genotype in the mouse modifies cytotoxic and proliferative effects seen in urinary bladders of wild type mice after exposure to inorganic arsenic. Female wild type C57BL/6 mice and As3mt KO mice were divided into 3 groups each (n=8) with free access to a diet containing 0, 100 or 150 ppm of arsenic as arsenite (As(III)). During the first week of As(III) exposure, As3mt KO mice exhibited severe and lethal systemic toxicity. At termination, urinary bladders of both As3mt KO and wild type mice showed hyperplasia by light microscopy. As expected, arsenic-containing granules were found in the superficial urothelial layer of wild type mice. In As3mt KO mice these granules were present in all layers of the bladder epithelium and were more abundant and larger than in wild type mice. Scanning electron microscopy of the bladder urothelium of As3mt KO mice treated with 100 ppm As(III) showed extensive superficial necrosis and hyperplastic changes. In As3mt KO mice, livers showed severe acute inflammatory changes and spleen size and lymphoid areas were decreased compared with wild type mice. Thus, diminished arsenic methylation in As3mt KO mice exacerbates systemic toxicity and the effects of As(III) on the bladder epithelium, showing that altered kinetic and dynamic behavior of arsenic can affect its toxicity.