A review of the toxicology of acrylamide.

Acrylamide (ACR) is a chemical used in many industries around the world and more recently was found to form naturally in foods cooked at high temperatures. Acrylamide was shown to be a neurotoxicant, reproductive toxicant, and carcinogen in animal species. Only the neurotoxic effects were observed in humans and only at high levels of exposure in occupational settings. The mechanism underlying neurotoxic effects of ACR may be basic to the other toxic effects seen in animals. This mechanism involves interference with the kinesin-related motor proteins in nerve cells or with fusion proteins in the formation of vesicles at the nerve terminus and eventual cell death. Neurotoxicity and resulting behavioral changes can affect reproductive performance of ACR-exposed laboratory animals with resulting decreased reproductive performance. Further, the kinesin motor proteins are important in sperm motility, which could alter reproduction parameters. Effects on kinesin proteins could also explain some of the genotoxic effects on ACR. These proteins form the spindle fibers in the nucleus that function in the separation of chromosomes during cell division. This could explain the clastogenic effects of the chemical noted in a number of tests for genotoxicity and assays for germ cell damage. Other mechanisms underlying ACR-induced carcinogenesis or nerve toxicity are likely related to an affinity for sulfhydryl groups on proteins. Binding of the sulfhydryl groups could inactive proteins/enzymes involved in DNA repair and other critical cell functions. Direct interaction with DNA may or may not be a major mechanism for cancer induction in animals. The DNA adducts that form do not correlate with tumor sites and ACR is mostly negative in gene mutation assays except at high doses that may not be achievable in the diet. All epidemiologic studies fail to show any increased risk of cancer from either high-level occupational exposure or the low levels found in the diet. In fact, two of the epidemiologic studies show a decrease in cancer of the large bowel. A number of risk assessment studies were performed to estimate increased cancer risk. The results of these studies are highly variable depending on the model. There is universal consensus among international food safety groups in all countries that examined the issue of ACR in the diet that not enough information is available at this time to make informed decisions on which to base any regulatory action. Too little is known about levels of this chemical in different foods and the potential risk from dietary exposure. Avoidance of foods containing ACR would result in worse health issues from an unbalanced diet or pathogens from under cooked foods. There is some consensus that low levels of ACR in the diet are not a concern for neurotoxicity or reproductive toxicity in humans, although further research is need to study the long-term, low-level cumulative effects on the nervous system. Any relationship to cancer risk from dietary exposure is hypothetical at this point and awaits more definitive studies.

A sensitive delayed-type hypersensitivity model in the rat for assessing in vivo cell-mediated immunity.

Many drugs and other chemicals can alter cell-mediated immunity (CMI), a response that often correlates with delayed-type hypersensitivity (DTH). Several DTH assays were evaluated to determine a method best suited for assessing chemically induced modulation of CMI in rats. The effects of various antigens, adjuvants, doses, routes, and immunosuppressants were investigated. The DTH method which produced optimum results in rats uses a footpad swelling reaction elicited by specific preparations of bovine serum albumin (BSA) and Freund's complete adjuvant (FCA). The rats were sensitized with 100 micrograms BSA in FCA injected subcutaneously at the base of the tail, and were challenged 7 days later with 75 microliter of 2% heat-aggregated BSA suspension injected into the left rear footpad. Footpad swelling was measured with pressure calipers 24 h later and compared to the contralateral footpad which was sham-injected with 75 microliters of physiological saline. Antigen-injected footpads were nearly double the thickness (7-8 mm) of the control footpads (3-4 mm), and variation between animals was small (CV = 5%). Dexamethasone and cyclophosphamide significantly suppressed the DTH reaction. Histopathological examination of the DTH reaction sites revealed a mononuclear cell infiltrate which is characteristic of type IV hypersensitivity. In addition to being highly quantitative and sensitive, this method provides a simple and reproducible assessment of CMI responses in the rat.

Alteration of natural killer cell-mediated cytotoxicity in rats treated with selenium, diethylnitrosamine and ethylnitrosourea.

Weanling, female Sprague--Dawley rats were divided into 14 separate groups. Three of these groups were administered 0.5, 2.0 or 5.0 ppm selenium (Se) in the drinking water for 10 weeks. Three groups received intraperitoneal injections of 1, 5 or 10 mg/kg diethylnitrosamine (DEN) twice weekly for 10 weeks. The remaining animals received 0.150% or 0.316% ethylurea (EU) in the feed and 1 or 10 ppm nitrite as sodium nitrite in the drinking water either alone or in combination. Separate groups of rats treated with cyclophosphamide (CY) were included as positive immuno-suppressed controls. Following the 10-week chemical exposure period, splenic natural killer (NK) cell-mediated cytotoxicity was assessed by a 4-h chromium release assay using YAC-1 tumor cells as targets. The NK cell cytotoxic response was enhanced in both the low and medium dose selenium-exposed groups. In contrast, rats exposed to 0.316% EU + 10 ppm NO2 had significantly depressed NK cell activity. CY treatment also resulted in a significant reduction of splenic NK cell cytotoxicity.

Increased susceptibility of adult rats to azoxymethane-induced aberrant crypt foci.

The purpose of this study was to compare azoxymethane-induced aberrant crypt foci development in the colons of young and adult rats. Young (4 weeks of age) and adult (50 weeks of age) Sprague-Dawley rats were treated with two weekly injections of azoxymethane or saline. Rats were killed either 6 or 14 weeks following the first injection, and the number, size and location of aberrant crypt foci were determined. At both the 6- and 14-week time points, the number of aberrant crypt foci in older rats was significantly greater than in young rats (P<0.01). A higher percentage of aberrant crypt foci were found in the region from the mid-colon to the cecum in older rats as compared to young rats. Colonic cell proliferation was evaluated using bromodeoxyuridine immunohistochemistry. Colonic cell proliferation indices in the rectal, mid-colon and cecal regions of young and older rats were similar in young compared to adult rats. Ten large ACF from each group were screened for mutations in the beta-catenin gene (Ctnnb1) by PCR single strand conformation polymorphism. No mutations were detected. These results demonstrate that older female rats are more susceptible to the induction of aberrant crypt foci by azoxymethane than young female rats. Differences in colonic cell proliferation or beta-catenin mutations in these two age groups do not appear to be responsible for differences in aberrant crypt foci development.

Effects of transplacental exposure to chlorinated phenols.

Female rats were exposed to 0, 5, 50 or 500 ppm of 2-chlorophenol (2-CP) or pentachlorophenol (PCP). The study was designed to produce progeny which were exposed to the chlorophenolic compounds both prenatally and postnatally. Percent conception, litter size, birth weight, and number of stillbirths was determined at parturition. Hematologic parameters and body weights of the progeny were recorded at weaning age (3 weeks). Effects on reproduction were observed in both the 2-CP and PCP-exposed groups, as indicated by decreased litter sizes and increased number of stillborn. The data indicate that these chlorinated phenolic compounds may be feto- or embryotoxic at high doses. Effects on hematologic parameters were not observed. Further study involving transplacental and chronic exposures to these chlorophenolic compounds appears warranted.

Trichloroacetic acid effects on rat liver peroxisomes and enzyme-altered foci.

The initiating and promoting effects of trichloroacetic acid (TCA) were investigated using a rat hepatic enzyme-altered foci bioassay. The experimental protocol used has been shown to induce gamma-glutamyltranspeptidase (GGT)-positive foci in hepatic tissue following an initiating dose with a genotoxic carcinogen. Twenty-four hours following 2/3 partial hepatectomy, rats received either a single oral dose (1500 mg/kg) or 5000 ppm TCA in drinking water for 10, 20, or 30 days. Two weeks after the end of TCA exposure, the rats were promoted for 3 or 6 months with 500 ppm phenobarbital in drinking water. TCA failed to induce GGT-positive foci using this initiation protocol. In addition, groups of 2/3 partially hepatectomized rats were initiated with a single oral dose of diethylnitrosamine (10 mg/kg) and then administered 50, 500, or 5000 ppm TCA drinking water. In this promotion protocol, TCA exposure resulted in a significant increase in the number of GGT-positive foci. The ability of TCA to stimulate peroxisomal-dependent palmitoyl-coenzyme A oxidation was also investigated. Only the 5000 ppm TCA treatment within the promotion protocol resulted in a significant, although minor, stimulation of peroxisomal enzyme activity. The findings support the hypothesis that TCA may possess weak promoting activity in the rat liver.

Characterization of a chemically induced tumor model and the effects of natural killer cell depletion by antiasialo GM-1.

A tumor model in the Sprague-Dawley rat has been developed and characterized in our laboratory using the polycyclic aromatic hydrocarbon 3-methylcholanthrene (3MC). Interactions between the tumorigenic process and the natural killer cell (NK) response were investigated in this study. Rats given a single injection of 1.5 mg 3MC had reduced NK activity the first three weeks after injection when compared to vehicle treated controls. Tumor incidence in this group reached 45% and 76% 12 and 20 weeks, respectively, after the 3MC injection. Cell lines were established from six of these tumors and were tested for in vitro lysis by NK cells. Sensitivity of these cells ranged from 2.9 to 12.2% compared to 32.3 to 37.5% for the NK sensitive YAC-1 target cells. Rabbit antiasialo GM-1 antibody (ASGM-1) was used to effect in vivo reductions of NK function at arbitrarily selected times after 3MC injection. Tumor incidence in the group of rats treated to reduce NK activity at the time of initiation (0-2 weeks) reached 100% in 20 weeks compared to 67% in the companion 3MC treated controls. The number of days to tumor was also decreased from 93 to 77 days in this group. Rats treated to reduce NK activity at other times (5-7 weeks or 10-12 weeks) did not have alterations in tumor incidence or latency that were different from controls. The study supports a role for NK cells in the early detection and removal of transformed cells and points out the dangers of transient immunosuppression.

Effect of cyclosporine on 3-methylcholanthrene-induced carcinogenesis and immune responses in the rat.

Cyclosporine A (CsA) was used to immunosuppress male Sprague-Dawley rats treated with the chemical carcinogen 3-methylcholanthrene (3MC). Rats treated with low doses of CsA (2.5 or 5 mg/kg) given 2 days prior to an injection of 3MC, and then daily for 2 weeks or twice weekly for 10 weeks did not develop tumors. Rats treated with 2.5 mg/kg CsA for 2 weeks beginning 5 days after a single 3MC injection had tumor incidence similar to rats treated with 3MC only. To further examine the effects of CsA on immune function, groups of rats were then treated with 2.5, 5, 10 or 20 mg/kg CsA daily for 14 days and immune function assessed by measuring delayed-type hypersensitivity (DTH), natural killer cell (NK) activity, and production of interleukin 2 (IL-2), interferon (IFN), prostaglandin E2 (PGE2) and specific IgG antibody. Natural killer cell cytotoxicity was enhanced and antibody production was suppressed in rats treated with all doses of CsA tested. Interleukin 2 production was elevated at the two lower doses, but antibody production, DTH reactions and synthesis of IL-2 and IFN were suppressed with the higher dose treatments (10, 20 mg/kg CsA). The enhanced NK activity seen in rats treated with the lower doses of CsA may be due to the increase in IL-2 production, while enhancement of NK activity at higher doses may be due to other mechanisms. The tumor data suggest that CsA does not prevent tumor formation in our chemical-induced model due to an increase in NK activity, since this enhancement was seen even when tumors did develop normally.

Subchronic 90 day toxicity of dichloroacetic and trichloroacetic acid in rats.

Male Sprague-Dawley rats were treated with either dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in the drinking water at levels of 0, 50, 500 and 5000 ppm for a period of 90 days to determine the toxicities associated with subchronic exposure. All animals were sacrificed and examined for gross and histopathologic lesions, serochemical changes, immune dysfunction, hepatic peroxisomal and mixed function oxidase enzyme induction and organ-body weight changes. Animals treated with DCA had decreased body weight gains (500 and 5000 ppm) and decreased total serum protein (all doses). Rats given either TCA (5000 ppm) or DCA (500 or 5000 ppm) had increased liver and kidney organ to body weight ratios. Rats offered DCA had significantly elevated alkaline phosphatase (500 and 5000 ppm) and alanine-amino transferase (5000 ppm). No consistent immunotoxicity was observed in animals exposed to either compound. Rats treated with 5000 ppm TCA or DCA had significantly increased hepatic peroxisomal beta-oxidation activity. These data, along with histopathologic changes, suggest that TCA and DCA produce substantial systemic organ toxicity to the liver and kidney during a 90-day subchronic exposure, although only at doses greater than those expected to occur in the environment.

Immunotoxicologic evaluation of chlorine-based drinking water disinfectants, sodium hypochlorite and monochloramine.

Male Sprague-Dawley rats were exposed to chlorine-based disinfectants in the drinking water from weaning to 12 weeks of age, at which time they were terminated and assessed for immune competence. Chlorine-based drinking water disinfectants used were sodium hypochlorite (5, 15 and 30 ppm) and monochloramine (9, 19 and 38 ppm). Parameters of immunity measured were spleen and thymus weights, antibody production, delayed-type hypersensitivity (DTH) reactions, natural killer cell (NKC) cytotoxicity, oxidative metabolism response (i.e chemiluminescence-CL) and phagocytosis by macrophages, and production of 2 immunoregulatory cytokines, interleukin 2 (IL2) and prostaglandin E2 (PGE2). Significant (P less than or equal to 0.05) reductions of spleen weight, DTH reactions, and oxidative metabolism by macrophages were observed only in groups of rats exposed to high levels (30 ppm) of sodium hypochlorite, while PGE2 production was elevated. Rats exposed to the higher doses of monochloramine had reduced spleen weights (38 ppm), decreased antibody synthesis (9 and 19 ppm) and augmented PGE2 production (19 and 38 ppm). These results extend the earlier observations of others that macrophage function of laboratory rodents may be impaired by exposure to high concentrations of chlorinated drinking water. Furthermore, the function of other major populations of immunocytes and types of immune responses may also be altered following subchronic exposure to high concentrations of chlorinated drinking water. These types of effects on the immune system are a previously unrecognized potential side-effect of the ubiquitous practice of disinfection of water with chlorine compounds. Alteration of immune function of chlorine-based disinfectant-exposed rats in this study was only evident at relatively high doses, and only selected immune responses were altered. It appears, therefore, that these chlorine-based disinfectants are not particularly strong immunodepressants. However, further studies in different species may be warranted in order to better extrapolate to implications to human health following chronic low-level exposure.

Cyclophosphamide-conditioned suppression of the natural killer cell response in rats.

Injection of rats with cyclophosphamide (CY) after their consumption of a novel saccharin-flavored drinking solution resulted in a conditioned aversion to saccharin and a conditioned suppression of natural killer cell (NKC) cytotoxicity. In this study, male Sprague-Dawley rats were conditioned by pairing saccharin with 50 mg/kg CY, an immunosuppressive drug with noxious gastrointestinal side-effects. Twenty-two and 26 days later, re-exposure of conditioned animals to saccharin alone re-enlisted the immunosuppressive effects of CY when NKC cytotoxicity was measured on day 29. Although CY also suppressed spleen cell number, IgG antibody titers and interleukin 2 (IL2) production, these immune responses did not appear to be affected by the behavioral conditioning paradigm in this experiment. Unique aspects of this study include the ability to measure multiple immune responses in a single rat and the finding that previous reports of behaviorally conditioned immunosuppression can be extended to another parameter, NKC cytotoxicity. These findings could have significant implications to human medicine, especially in the area of autoimmunity and immunodeficiency, and intervention and treatment of cancer.

Tissue residues, pathology and viral-induced mortality in mice chronically exposed to different cadmium salts.

Male Swiss Webster mice were exposed to one of three different cadmium salts (sulfate, acetate and chloride) in the drinking water for 10 weeks and then inoculated with a 14-day LD75 or LD35 dilution of encephalomyocarditis virus. the incidence of viral-induced mortality was lower in 17 of the 18 cadmium-treated groups of mice as compared to controls. Cadmium tissue residues and histopathologic lesions were essentially similar at comparable doses in all groups of mice regardless of the form of cadmium in the diet.

Effects of sphingomyelin on aberrant colonic crypt foci development, colon crypt cell proliferation and immune function in an aging rat tumor model.

Sphingomyelin (SPM) was assessed in older rats for in vivo effects on multiple immune responses and the development of colon preneoplastic lesions. Fifty-four-week-old rats were injected with 10 mg/kg body weight of the carcinogen azoxymethane (AOM), and then treated with 35 mg/kg body weight SPM orally for 6 weeks beginning 6 weeks after AOM treatment. None of the immune functions tested (antibody formation, delayed-type hypersensitivity or natural killer cell cytotoxicity) were significantly affected by SPM treatment. Natural killer (NK) cell activity was, however, decreased in all rats that were treated with AOM. There was a tendency for decreased aberrant crypt foci (ACF) numbers in the SPM-treated rats but this reduction was only significant for the largest lesions (> nine crypts per foci). The decreased ACF numbers were most evident in the proximal end of the colon. Colonic crypt cell proliferation was also decreased in SPM treated rats. This reduction was primarily in the base of the crypt column. Also, low numbers of ACF developed spontaneously in rats not treated with AOM, but no ACF were present in non-AOM rats that also received SPM. It appears that SPM may have effects on the post-initiation development of preneoplastic lesions in the rat colon but not on the immune functions assessed in this study.

Dietary indole-3-carbinol alters immune functions in rats.

To further elucidate the physiological mechanisms that may contribute to the dichotomy of effect of indole-3-carbinol (I3C) on cancer development, we examined immune functions representative of the three major branches of the immune system in rats fed either a high (150 mg/kg) or low (50 mg/kg) dose of I3C. Animals fed the high dose of I3C daily for 7 wk had significantly reduced natural killer cell activity. In contrast, T-cell-mediated delayed-type hypersensitivity was significantly elevated. Antibody production in response to the antigen keyhole limpet hemocyanin was not significantly altered compared to controls. These results indicate that exposure to I3C may have differential effects on major immune responses. The significance of these immune function alterations in tumor development will require additional investigation of the effects of dietary I3C on immune functions in appropriate tumor models.

Effect of dietary chlorogenic acid on multiple immune functions and formation of aberrant crypt foci in rats.

Adult male Sprague-Dawley rats were fed 70 mg/kg body weight chlorogenic acid (CHA) for 7 wk. One CHA-fed group was also given 2 injections of the colon carcinogen azoxymethane (AZO) on d 2 and 9 of CHA treatment. Three major types of immune responses were assessed: antibody production, specific cell-mediated immunity, and nonspecific cell-mediated immunity. The formation of AZO-induced aberrant crypt foci (ACF) in the colon were observed, as was colonic cell proliferation. There were no significant effects of CHA treatment on any of the immune parameters examined or on formation of preneoplastic lesions or cell proliferation in the colon. The overall nonsignificant trends in immune function, colon cell proliferation, and ACF development were, however, more consistent with immunosuppression and enhanced preneoplasia.

Effects of indole-3-carbinol on immune responses, aberrant crypt foci, and colonic crypt cell proliferation in rats.

Male Sprague-Dawley rats were treated orally with indole-3-carbinol (13C) for 7 wk at levels of 150, 100, and 50 mg/kg body weight. The rats were injected with 10 mg/kg body weight of the colon carcinogen, azoxymethane (AOM) on d 2 and 9 of 13C treatment. At termination of the study, all rats were assessed for immune function (humoral immunity, specific cell-mediated immunity, and nonspecific cell-mediated immunity). Colonic tissue was collected and examined for the presence of aberrant crypt foci (ACF) and proliferation of crypt cells. Antibody responses to antigen challenge were significantly suppressed in the animals exposed to the high dose of 13C. Delayed-type hypersensitivity responses, natural killer cell activity, the number and multiplicity of ACF, and cell proliferation parameters were not significantly different from those of the controls. Therefore, there was no clear protective or enhancing effect of 13C on ACF numbers or colonic cell proliferation indices. There was no strong correlation between changes in immune responses and the preneoplastic biomarkers of colon cancer.

The two faces of selenium-deficiency and toxicity--are similar in animals and man.

The purpose of this review article is to demonstrate the close parallelism of daily requirements, biological activity and minimum and maximum tolerable levels of selenium for animals and man. In addition, the carcinogenic/anticarcinogenic properties of selenium are discussed and a postulate of how these dichotomous effects may occur in accordance with selenium-induced immunomodulation is presented. A review of pertinent literature pertaining to the biological action of selenium in animals and man, including deficiency, toxicity, carcinogenicity and effects on immunity, is included to support these concepts. The predominant biochemical action of selenium in both animals and man is to serve as an antioxidant via the selenium-dependent enzyme, glutathione peroxidase, and thus protect cellular membranes and organelles from peroxidative damage. The signs and symptoms of selenium deficiency closely simulate each other for animals and man. Severe deficiency is characterized by cardiomyopathy while moderate deficiency results in less severe, myodegenerative syndromes such as muscular weakness and pain as well as a variety of other selenium-associated diseases. Clinical manifestations of many of these disorders require contributory factors, such as stress, to precipitate symptoms which are documented for animals and implicated for humans. Current evidence suggests that a daily selenium consumption for man of approximately 30 micrograms is necessary to prevent the selenium-deficient syndrome, Keshan disease, while approximately 90 micrograms/day/adult should be the minimum daily requirement for optimum biological performance. Recognizing that humans in several countries do not meet the proposed minimum daily requirement of 90 micrograms, several compelling reasons are presented in deriving this minimal daily nutritional intake. Selenosis can occur in laboratory animals, livestock, and humans following long-term exposure to selenium concentrations as low as 5 mg selenium/kg of diet (5 ppm). The selenium-induced lesions for all species are similar, which once again illustrates a positive corollary for selenium effects in both animals and man. From compilation of available data, the maximum tolerable level for selenium in man could be considered in the range of 1000 to 1500 micrograms/day. This is in contrast to the currently recommended maximum human tolerable level of 500 micrograms/day. The amount of selenium that can be tolerated, however, is dependent upon individual biological variation, nutritional status and general state of health.(ABSTRACT TRUNCATED AT 400 WORDS)

Lead-cadmium interaction: effects on viral-induced mortality and tissue residues in mice.

Simultaneous exposure to lead and cadmium is highly probable due to common environmental sources of exposure. Mice were exposed simultaneously for 10 wek to lead and cadmium and effects were observed on accumulation of the metals in tissues and susceptibility to viral challenge. The incidence of viral-induced mortality was enhanced in all groups of mice exposed to lead and suppressed in groups of mice treated with cadmium. Mice exposed to both lead and cadmium had mortality rates intermediate to those observed after exposure to the individual metals. Lead and cadmium residues in tissues of mice exposed to both metals, as compared to residues in tissues of mice which received lead or cadmium only, indciated that interaction occurs in regard to metabolism, storage, and excretion of these elements. Chronic coexposure to lead and cadmium appears to alter certain effects produced by exposure to each metal alone.

Polycythemia produced in rats by environmental contaminants.

Polycythemia developed in progeny from mothers who were exposed during pregnancy to a combination of methylmercury chloride plus ethylurea and sodium nitrite. The polycythemia occurred as early as one month of age and as many as 24% of the offspring developed the polycythemic condition. Many features of this condition are similar to those of polycythemia vera in man, such as elevated hematocrits and white and red blood cell counts, splenomegaly, and hyperplasia of bone marrow accompanied by megakaryocytosis.

Antibody suppression by cadmium.

Mice exposed to subclinical doses of cadmium chloride for ten weeks and inoculated with antigen six weeks after discontinuance of exposure had a remarkable decrease in antibody-forming cells, particularly IgG. These results indicate that immunosuppression produced by environmental contaminants may persist for several months after exposure to the contaminant.