Katherine S. Squibb

Immunotoxicity of low level cadmium exposure in fish : an alternative animal model for immunotoxicological studies

Cadmium represents a major aquatic pollutant in many parts of the world. Yet, despite the fact that cadmium accumulates in high concentrations in fish tissues, is found in polluted aquatic environments, and is carcinogenic and immunotoxic in a variety of mammalian species, the effects of cadmium on the immune responses of directly exposed aquatic species have not been clearly defined. This study was designed to assess the effects of in vivo cadmium exposure, at a concentration found in contaminated aquatic environments, on the immune defense mechanisms of fish. In this study, no effects were observed upon body weight, lysozyme activity, of cell viability, despite the high concentration of accumulated cadmium in the gills and liver. Furthermore, in the absence of any clinical manifestations or overt toxicity, exposure of rainbow trout to waterborne cadmium at 2 ppb altered macrophage-mediated immune functions, including phagocytosis and free radical production, in a time-dependent manner. Similar immunotoxic effects of cadmium have also been observed in mammals. Although interspecies comparisons between mammalian and fish immune responses are extremely complicated and need to be approached with caution, results from this study suggest the applicability of fish as an additional/alternative animal model for immunotoxicological studies.

DIFFERENTIAL DNA-PROTEIN CROSSLINKING IN LYMPHOCYTES AND LIVER FOLLOWING CHRONIC DRINKING WATER EXPOSURE OF RATS TO POTASSIUM CHROMATE

Carcinogenic chromium (VI) compounds are persistent environmental contaminants with potential for human exposure through drinking water. One lesion associated with chromium (VI) exposure is the formation of DNA-protein crosslinks (DPC). In an attempt to develop markers of chromium exposure, the formation of DPC in lymphocytes was investigated. Fisher 344 rats were exposed to K2CrO4 in their drinking water for 3 and 6 weeks at concentrations of 100 and 200 ppm chromium. No DPC could be detected in isolated splenic lymphocytes using the alkaline elution technique or by using a polyclonal antibody to chromate-induced DPC. However, increased complexing of proteins with DNA was demonstrated in liver following 3 weeks of exposure at both 100 and 200 ppm chromium. Intraperitoneal administration of potassium chromate did not induce detectable DPC in lymphocytes; however, an increased association of proteins with isolated DNA was detected in the liver. DPC were also induced in isolated splenic lymphocytes following a 2-hr exposure in vitro to 100 microM K2CrO4 in a salts-glucose medium. Although chromium was detected in blood, liver, and kidney, blood levels were comparatively much lower. A comparison of chromium levels required to induce DPC in lymphocytes in vitro and the amount absorbed orally suggests that the white blood cell chromium levels following oral exposure may be too low to induce measurable DNA-protein crosslinks in lymphocytes.

Distribution of chromium within cells of the blood

Although a number of investigators have examined the uptake of chromium in red blood cells (RBC) or whole blood, little is known about chromium uptake in white blood cells (WBC). Radiolabeled chromium (51Cr) was used to determine chromium uptake and distribution. Isolated RBC and enriched WBC populations were exposed in vitro to potassium chromate (Cr+6) and uptake was determined over a 2-hr time period. Exposure of either rat or human blood cells to 50 microM K2CrO4 for 2 hr resulted in greater accumulation of chromium within WBC than RBC. Uptake by rat WBC was significantly greater than that of human; whereas, uptake by human RBC was greater than that of the rat. Exposure of human whole blood to 50 microM K2CrO4, prior to isolation of WBC, also resulted in an increased uptake of chromium by WBC. Fisher 344 rats were exposed either orally or intravenously to a single dose of K2CrO4 and the distribution of chromium within blood cells was determined 1 hr, 24 hr, or 7 days following exposure. Regardless of the route or time following exposure, WBC chromium levels were consistently greater than those of RBC. However, the absolute levels of chromium did change with time. A comparison of chromium distribution 24 hr following a single oral exposure (1 ppm Cr+6) to the distribution 7 days following exposure demonstrated a reduction in chromium levels for RBC (10-fold) and for WBC (approximately 2.5-fold). In contrast, intravenous administration of chromate resulted in no significant decrease in RBC chromium levels when compared 1 hr, 24 hr, and 7 days following exposure. Although no difference in WBC chromium content was observed at 1 and 24 hr after exposure, an approximate 1.7-fold decrease in chromium content was detected at Day 7 for WBC. Intravenous administration of chromic chloride (Cr+3) resulted in a low level of chromium associated with RBC following 1 hr, and chromium was undetected in the WBC. These data demonstrate that WBC accumulate hexavalent chromium following both in vitro and in vivo exposure. In addition, white blood cells accumulate chromium to a greater extent than red blood cells. Since WBC accumulate chromium, their use as a target for the development of biomarkers of chromium exposure may be warranted.

Effects of prior exposure history on cytochrome P4501A mRNA induction by PCB congener 77 in atlantic Tomcod

Abstract Levels of P450IA mRNA in tomcod collected from the Miramichi River, Canda and the Hudson River, New York were measured by slot blot analyses after the fish had been depurated and i.p. injected with Aroclor 1254, PCB congener 77, or PCB congener 105. Elevated expression of P450IA mRNA was observed in congener 77-treated Miramichi tomcod, whereas no induction was seen in the Hudson River fish. Tomcod from both rivers were induced by injection with beta-naphthoflavone. These results suggest that the prior exposure history of the Hudson River tomcod may have affected their non-inducibility by treatment with PCB congener 77.

Development of fish peritoneal macrophages as a model for higher vertebrates in immunotoxicological studies: I. Characterization of trout macrophage morphological, functional, and biochemical properties

The immune defense mechanisms of fish are not as well characterized as those of mammals but seem to be related and similarly competent. Because of this, there is an increased interest in the immune responses of fish as models for higher vertebrates in immunotoxicological studies. Prior to such studies, baseline criteria for specific components of the immune response needed to be established. For this study, we have examined trout macrophage morphology using light and scanning electron microscopy, phagocytic activity, random and stimulus-directed migration, and superoxide anion radical (O2-) production for resident and lipopolysacharide (LPS) or Aeromonas salmonicidae-elicited rainbow trout (Oncorhynchus mykiss) peritoneal macrophages (M phi). Following peritoneal lavage, greater than 89% of the cells were M phi as determined by differential counts and nonspecific esterase staining. Immunization with LPS and A. salmonicidae increased M phi number approximately 5 and 13-fold, respectively, and overall size. Trout M phi were phagocytically active engulfing serum opsonized latex particles and were mobile, migrating both randomly and in a directed fashion towards formyl-methionine-L-leucine-L-phenylalanine (FMLP) and trout serum-derived complement fragment C5a. Concentrations of FMLP (100 nM) and C5a (0.01-1%) effective for attracting trout M phi are the same as those used to attract rabbit M phi. Resident trout M phi produced negligable quantities of .O2- following stimulation with 1 micrograms/ml phorbol myristate acetate; Aeromonas-elicited M phi produced .O2- in a time-dependent manner which peaked after 60 min at 2.9 nmol per 2 x 10(5) cells and then declined. The results of this study provide a data base for future toxicological studies with trout peritoneal M phi and indicate the usefulness of this system for immunotoxicological studies.

Cytochrome P450IA mRNA expression in feral hudson river tomcod

We sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, we found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached by 5 days. Intraperitoneal injection of beta-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers.

Detection of cadmium exposure in rats by induction of lymphocyte metallothionein gene expression

The induction of metallothionein (MT) gene expression in lymphocytes of rats was determined in order to detect exposure in vivo to cadmium. Both acute and chronic CdCl2 exposures resulted in the induction of the MT-1 gene in lymphocytes as measured by standard RNA Northern blot analysis. Twenty-four hours following an ip injection of 3.4 mg/kg CdCl2, a ninefold increase in MT gene expression was observed in lymphocytes, as well as five- and sevenfold increases in liver and kidney, respectively. Oral exposure of rats to 1-100 ppm CdCl2 via drinking water resulted in an approximate twofold enhanced MT signal in lymphocytes after 6 wk, and a threefold increase after 13 wk of exposure to 100 ppm Cd. No increases in lymphocyte MT gene expression were observed after 3 wk of Cd exposure. Liver MT gene expression was substantially induced following chronic Cd exposure, while kidney was not, although this organ had a higher basal expression of the MT-1 gene. Analysis of tissue Cd burdens demonstrated a dose-response Cd accumulation in liver and kidney, but only kidney burdens increased substantially with prolonged Cd exposure. These results demonstrate the utility of lymphocyte gene expression assays to detect in vivo toxicant exposure, and thus their applicability as molecular biomarker assays for human exposure assessment.

Comparison of the uptake and distribution of chromate in rats and mice

The purpose of this study was to evaluate species differences in tissue accumulation of chromium. Rats and mice were orally exposed to Cr(VI) (potassium chromate) via drinking water (8 mg/d/kg body wt for 4 or 8 wk) or by ip injection (0.3 and 0.8 mg/d/kg, for 4 or 14 d). Chromium concentrations were measured by atomic absorption spectrophotometry, and tissues were compared for exposure route and species differences. After oral exposure, irrespective of treatment duration, liver concentrations of chromium were three to four times higher in mice than rats, whereas kidney concentrations were about 50% lower. However, after ip injection, kidney and blood concentrations in rats were two- and four-fold, higher, respectively. Both rats and mice showed high values of Cr concentration in the bone. After single ip injection of Na251CrO4; Cr concentrations were higher in the blood of rats than mice both after 24 and 72 h. Red blood cell concentrations of Cr were also greater in rats than mice by approximately threefold, whereas white blood cell Cr concentrations were higher in mice than rats. There was also a twofold greater binding of Cr/μmol of hemoglobin in rats compared to mice. These data indicate that species differences exist for Cr metabolism and that they differ with respect to the route of exposure. These results may be owing to species differences in the reduction of Cr and different binding of Cr to hemoglobin.

Fate of 7,12-dimethylbenz(a)anthracene in rainbow trout,Salmo gairdneri

Polycyclic aromatic hydrocarbons (PAH) are contaminants of surface waters and sediments, especially near urban centers. Although aquatic biota accumulate PAHs from environmental sources, metabolism may be rapid, and biota sampled from contaminated areas often have concentrations lower than might be estimated from bioconcentration factors. In some cases PAH metabolism by aquatic biota may create reactive intermediates, some of which have been related to chronic effects in fishes. This report describes the fate and distribution of 7,12-dimethylbenz(a)anthracene (DMBA) after oral administration to rainbows trout (Salmo gairdneri). Emphasis has been placed on the disposition of DMBA among tissues and on DMBA transformation in the hepatobiliary system.

In vivo kinetics of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene assimilation and metabolism in rainbow trout

Abstract Benzo( a )pyrene (BaP) and 7,12-dimethylbenz( a )anthracene (DMBA) are polycyclic aromatic hydrocarbons (PAH) that occur in a variety of environmental media. BaP and DMBA are indirect-acting mutagens and carcinogens, and both induce neoplasia in fishes. 1,2 BaP has been used to study PAH-induction of mixed function oxidase systems and PAH metabolism in fishes; 3,4 studies of DMBA in fishes are limited to analyses of tissue disposition 5 and induction of drug-metabolizing enzymes. 6 We report here the results of comparative studies of BaP and DMBA assimilation, elimination, tissue disposition and metabolism in rainbow trout after administration of known doses of these contaminants via the digestive tract.