Robert M. Bare

Cadmium-induced DNA strand damage in cultured liver cells : reduction in cadmium genotoxicity following zinc pretreatment

It is well established that zinc can decrease the carcinogenicity and toxicity of cadmium. In some tissues this may be due to the induction of metallothionein (MT). Therefore, in the present investigation, the effect of zinc pretreatment on cadmium-induced DNA strand damage was determined. The alkaline elution technique was used to assess DNA single strand damage (SSD) in cultured cells derived from rat hepatocytes (TRL-1215), a cell line previously shown to have an active MT gene. The ability to detect SSD in TRL-1215 was established following exposure to gamma-irradiation. Exposure to increasing doses of gamma-irradiation (150-600 rad) resulted in a dose-dependent increase in SSD. Exposure of TRL-1215 cells to CdCl2 for 1 hr at 37 degrees C, using concentrations from 5 to 250 microM, failed to induce detectable SSD in these cells; however, exposure to 500 microM CdCl2 resulted in significant SSD. A time-dependent increase in SSD was demonstrated following a 2 hr continuous exposure to 500 microM CdCl2. Pretreatment of cells with 80 microM zinc acetate, 18 hr prior to exposure to 500 microM CdCl2, resulted in markedly reduced SSD when compared to non-pretreated cells. Zinc pretreatment increased the level of MT gene expression as well as MT protein production. The decrease in DNA strand damage associated with cadmium exposure was not due to a decrease in cadmium accumulation by zinc pretreated cells. In fact, cellular cadmium burden was increased over 2-fold following zinc pretreatment. In addition to protection against cadmium genotoxicity, zinc pretreatment also reduced the cytotoxicity associated with a 2-hr, 500 microM cadmium exposure. These data indicate that zinc pretreatment reduces cadmium genotoxicity, possibly through alterations in MT gene expression.

Enhanced metallothionein gene expression is associated with protection from cadmium‐induced genotoxicity in cultured rat liver cells

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that appear to play an important role in the cellular defense system against cadmium toxicity. Although substantial evidence exists demonstrating a reduction in cadmium toxicity concomitant with MT induction, little is known about the possible effects of stimulation of MT synthesis on cadmium-induced genotoxicity. Thus, the alkaline elution technique was used to assess single-strand DNA damage (SSD) in TRL-1215 cells, a liver-derived cell line shown to have inducible MT gene expression. The SSD accumulated over a 2-h time period in a time-dependent manner following exposure to 500 microM CdCl2. Low-concentration cadmium pretreatment (10 microM CdCl2, 24 h) provided protection against the genotoxicity of high-concentration cadmium (500 microM CdCl2, 2 h). A 2-h exposure to 500 microM CdCl2 had no effect on viability, as assessed using a tetrazolium-dye based assay, in cells from either the pretreated or nonpretreated group. Metallothionein was induced in a time-dependent manner by low-concentration cadmium pretreatment: Exposure for 24 and 48 h resulted in 3.3- and 6.4-fold increases, respectively. In addition, a 24-h exposure to low-concentration cadmium resulted in an increase in MT-I gene expression. Cadmium accumulation was 2.6-fold greater in low-concentration cadmium-pretreated cells as compared to nonpretreated cells. These data demonstrate that low-concentration cadmium pretreatment provides protection against cadmium-induced single-strand DNA damage and support the hypothesis that this protection is due to stimulation of MT gene expression.

Metallothionein gene expression in testicular interstitial cells and liver of rats treated with cadmium

The rodent testes are generally more susceptible to cadmium (Cd)-induced toxicity than the liver. Cd induces predominantly testicular interstitial cell (TIC) tumors. In order to clarify the molecular mechanism underlying tissue differences in Cd sensitivity, we compared Cd-induced metallothionein (MT) gene expression, MT protein accumulation, and Cd retention in freshly isolated TICs and liver. Adult male Fischer rats received a s.c. injection of 4.0 micromol Cd/kg or vehicle and 24 h later tissues were sampled and TICs isolated. MT-I and MT-II mRNA levels were determined by slot-blot analysis followed by densitometry scanning, and MT was estimated by the Cd-heme method. Testicular lesions were not grossly or histologically observed in rats treated with 4 micromol Cd/kg. Both MT mRNA and MT (as determined by Cd-binding capacity) were constitutively present in TICs as well as the liver. TICs isolated from Cd-treated rats accumulated more Cd (4-fold), and had higher levels of MT-I (1.9-fold) and MT-II (1.4-fold) mRNAs over control, but contained less MT (30% decrease) than TICs isolated from control animals. Cd exposure substantially increased hepatic Cd content (6000-fold), MT (58-fold), and MT-I mRNA (5.3-fold), but did not increase MT-II mRNA. Thus, our findings indicate that, although low-dose Cd exposure results in increases of MT mRNA in TICs it does not enhance MT synthesis within these cells. The inability to induce the metal-detoxicating MT-protein, in response to Cd, might account for higher susceptibility of testes to Cd toxicity and carcinogenesis relative to liver.

Anticarcinogenic effects of cadmium in B6C3F1 mouse liver and lung

The B6C3F1 mouse liver has been widely used for the evaluation of carcinogenic or tumor promoting efficacy of various organic compounds, although little is known about the actions of metallic carcinogens in this system. Thus, the ability of cadmium to initiate or promote tumors in B6C3F1 mouse liver was studied. In promotion studies, diethylnitrosamine (DEN; 90 mg/kg, ip) was given as an initiator to 5-week-old mice followed 2 weeks later by 500 or 1000 ppm of cadmium in drinking water for 50 weeks. DEN caused an elevation of liver tumor incidence (13 tumor bearing mice/45 total) over control (1/48) which was prevented by cadmium (DEN + 500 ppm cadmium, 3/42; DEN + 1000 cadmium, 0/47). Cadmium alone did not further reduce the very low spontaneous liver and lung tumor incidence at approximately 1 year of age. DEN-induced lung tumor incidence (15/45) was also reduced by cadmium (DEN + 500 ppm cadmium, 11/42; DEN + 1000 ppm cadmium, 1/47) to control levels (0/48). In initiation studies, cadmium (20 or 22.5 mumol/kg, sc) was given to 5-week-old mice (n = 30-60) 2 weeks before an established promoting regimen of sodium barbital (BB) in drinking water at 500 ppm level was begun. Barbital in drinking water was given continuously for up to 92 weeks. Such cadmium doses caused acute, focal hepatic necrosis. Mice treated with BB and killed at 97 weeks of age showed an elevation of liver tumor multiplicity (7.44 tumors/liver) over control (2.24) that was prevented by cadmium in a dose-related manner (20 mumol/kg cadmium + BB, 3.93; 22.5 mumol/kg cadmium + BB, 1.87). Cadmium alone given by injection also reduced spontaneous liver tumor multiplicity. These results indicate that cadmium inhibits tumor formation in the B6C3F1 mouse liver initiation/promotion system regardless of route of exposure or sequence of administration. The possibility exists that cadmium has a specific toxicity toward previously initiated cells within liver and lung.

Lack of correlation between the inducibility of metallothionein mRNA and metallothionein protein in cadmium-exposed rodents.

Cadmium (Cd) is carcinogenic in humans and laboratory animals. Depending on the duration and route of exposure, Cd can also induce damage in the liver, kidneys and lungs. In certain tissues, metallothionein (MT) proteins are induced by Cd exposure and associated with native and acquired tolerance to the metal. Rats are generally more sensitive than mice to Cd carcinogenicity; however, sensitivity can vary markedly between different strains of the same rodent species. To further define the role of MT in Cd toxicity and carcinogenesis, adult male Wistar rats and adult male C57 and DBA mice were treated with CdCl2 and liver, kidney, and lung were analyzed for Cd, MT mRNA, and MT protein 24 h later. Dose-related increases in Cd were detected in the livers and kidneys of all animals tested; however, increases in pulmonary Cd were observed only in C57 mice, and only at the highest CdCl2 dose. While hepatic Cd concentrations were similar in the rats and mice, renal Cd concentrations were similar in the rats and DBA mice but were nearly 2-fold higher in C57 mice at the highest CdCl2 dose. Dose-related increases in MT mRNA occurred in the livers and lungs of all animals tested. Hepatic MT mRNA concentrations were highest in the rats, and C57 mice exhibited the greatest magnitude of hepatic MT mRNA induction. Dose-related increases in renal MT mRNA were also detected in both strains of mice, but between the two strains, C57 mice exhibited substantially higher levels of renal MT mRNA induction. Basal levels of renal MT mRNA were higher in the rats than in the mice, and transcription of the MT gene was not inducible in the rat kidney at any of the CdCl2 doses used. In comparison, basal levels of pulmonary MT mRNA were similar in the rats and DBA mice, were substantially lower in C57 mice, and increases in pulmonary MT mRNA were most pronounced in the rats. Analysis of MT protein revealed dose-related increases in the livers and kidneys of all animals tested. C57 mice had the lowest basal and induced levels of hepatic MT, and basal levels of renal MT were much higher in the rats than in mice of either strain. Although dose-related increases in pulmonary MT were similar in both strains of mice, pulmonary MT levels were much lower and not inducible in the rats. Overall our experiments revealed complex profiles of Cd distribution and MT expression that varied between tissues, species and strains, and often did not directly correlate with sensitivity to damage. The results suggest that Cd distribution, inducibility of the MT gene, and levels of MT protein, must all the considered when predicting susceptibility to Cd toxicity and carcinogenicity at particular target sites.

Reduced Fhit protein expression in nickel-transformed mouse cells and in nickel-induced murine sarcomas.

Nickel compounds are carcinogenic and induce malignant transformation of cultured cells. Since nickel has low mutagenic potential, it may act predominantly through epigenetic mechanisms, including down-regulation of tumor suppressor genes. FHIT is a tumor suppressor gene whose expression is frequently reduced or lost in tumors and pre-malignant lesions. Previously, we have shown that the phosphohydrolase activity of Fhit protein, associated with its tumor suppressor action, is inhibited by nickel [12]. In cells, such effect would assist in carcinogenesis. The latter could be further enhanced if nickel also lowered cellular levels of Fhit protein itself, e.g. by down-regulation of FHIT gene. To test this possibility, we determined Fhit protein and Fhit-mRNA levels in a nickel-transformed mouse cell line and in nickel-induced murine sarcomas. In B200 cells, derived by nickel treatment of BALB/c-3T3 cells and exhibiting a malignant phenotype, Fhit protein levels were 50% of those in the parental cells, while Fhit-mRNA expression remained unchanged. A decrease of up to > 90percnt; in Fhit protein levels was also observed in 22 local sarcomas (mostly fibrosarcomas) induced by i.m. injection of nickel subsulfide in C57BL/6 and MT+ (C57BL/6 overexpressing metallothionein) mice, as compared with normal muscles. Moreover, Fhit was absent in 3 out of 10 sarcomas from MT+ mice and in 1 of 12 sarcomas from C57BL/6 mice. The lack of Fhit protein coincided with the absence of the Fhit-mRNA transcript in these tumors. However, in the other tumors, the decreased Fhit levels were not always accompanied by reduced expression of Fhit-mRNA. Thus, the observed lowering of Fhit protein levels is mostly associated with changes in mRNA expression and protein translation or turnover rates, and rarely with a full silencing of the gene itself. Overall, the decline of Fhit in cells or tissues malignantly transformed by nickel may indicate possible involvement of this effect in the mechanisms of nickel carcinogenesis.

Testosterone pretreatment mitigates cadmium toxicity in male C57 mice but not in C3H mice

Previous work has indicated that testosterone pretreatment protects against cadmium-induced toxicity in male rats while other data indicate that pretreatment of mice with testosterone offers no such protection against cadmium. Since cadmium toxicity may vary widely with species and strain, we examined the effect of testosterone pretreatment on cadmium toxicity in two strains of mice, one that is sensitive (C3H) and one that is resistant (C57) to cadmium toxicity. A single sc injection of 20 micromol CdCl2/kg to C3H mice or 45 micromol CdCl2/kg to C57 mice proved very toxic, causing 50%, and 44% mortalities, respectively. However, when C57 mice were pretreated with testosterone (5 mg/kg, s.c., at - 48, - 24, and 0 h) prior to cadmium (45 micromol/kg), complete resistance to cadmium-induced lethality developed. Testosterone had no effect on cadmium-induced lethality in C3H mice. Testosterone prevented extensive hepatocellular damage caused by cadmium in C57 mice and also significantly reduced cadmium-induced elevations in serum lactate dehydrogenase (LDH) activity and blood urea nitrogen (BUN), which are indicators of hepatic and renal function, respectively. The toxicokinetics of cadmium were apparently not affected by testosterone pretreatment, as the distribution of cadmium to liver in either strain was unchanged by the steroid. Cadmium-induced metallothionein (MT) levels in liver and kidney of C57 mice were increased in testosterone-pretreated mice given the higher doses of metal but no such enhancement of MT synthesis occurred in C3H mice. This increase in MT may provide some level of protection against cadmium toxicity in the C57 mice. These results indicate that testosterone pretreatment prevents toxicity of cadmium in male C57 mice, possibly through enhancement of MT synthesis, but has no effect in male C3H mice.