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.

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.

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.

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.

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.

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.

Cadmium carcinogenesis in male Wistar [Crl:(WI)BR] rats: dose-response analysis of effects of zinc on tumor induction in the prostate, in the testes, and at the injection site.

The ability of zinc acetate to modify the carcinogenic effects of CdCl2 in male Wistar [Crl:(WI)BR] rats was studied over a 2-year period. Groups of rats received a single s.c. injection of Cd (30.0 mumol/kg) in the dorsal thoracic midline or i.m. in the right thigh at time 0. Zinc was given in three separate s.c. doses of 0.1, 0.3, or 1.0 mmol/kg (at -6, 0, and +18 h relative to cadmium) in the lumbosacral area or p.o. at 100 ppm in the drinking water (-2 to +100 weeks). Cadmium treatments (s.c.) resulted in the appearance of tumors at the injection site and in the testes. The incidence of s.c. injection site tumors (mostly mixed sarcomas) was markedly reduced by high dose (1.0 mmol/kg) s.c. zinc (50% reduction) and was almost abolished by p.o. zinc (92% reduction). Testicular tumors (mostly Leydig cell adenomas) induced by s.c. cadmium were reduced in a dose-related fashion by zinc and were found to be highly dependent on the ability of zinc to prevent the chronic degenerative effects of cadmium in the testes. Oral zinc had no effect on s.c. cadmium-induced testicular tumors, while i.m. cadmium alone did not induce these tumors. In rats in which s.c. cadmium-induced testicular tumors and chronic degenerative effects were prevented by zinc (1.0 mmol/kg, s.c.), a marked elevation in prostatic tumors (exclusively adenomas) occurred (control, 9.6%; cadmium plus high zinc 29.6%). Cadmium given i.m., which did not result in testicular tumors or degeneration, also induced an elevated incidence (42.3%) of prostatic tumors, again indicating a dependence on testicular function. Prostatic tumor incidence was also significantly elevated (25.0%) in rats receiving 1.0 mmol/kg zinc, s.c., in combination with i.m. cadmium. These results indicate that zinc inhibition of cadmium carcinogenesis is a complex phenomenon, depending not only on dose and route but also on the target site in question.

In vitro polymerization of histones by carcinogenic nickel compounds.

This study was undertaken to explore whether nuclear chromatin constituents can participate in and/or be affected by redox reactions catalyzed by nickel, like those of nickel complexes with small peptides, e.g. tetraglycine (G4) and oxygen. Calf thymus DNA, nucleohistone (NH) or free histones were incubated at 37 degrees C, pH 7.6, for up to 96 h with nickel(II)acetate (NiAcet) or nickel subsulfide (Ni3S2) and/or G4. The effects on DNA and NH were studied by means of melting profiles. Free individual histones and histones extracted from NH prior to and after exposure to nickel compounds and/or G4 were examined by electrophoresis on polyacrylamide gels. Two-day exposure of DNA to NiAcet, G4, or NiAcet + G4 had no significant effect on its melting temperature. Incubation of NH with NiAcet, however, markedly increased its melting temperature by 2.2 +/- 0.3 degrees C after 24 h and 3.0 +/- 0.5 degrees C after 96 h (P less than 0.01 versus NH alone at either time). Incubation of NH with NiAcet + G4 also resulted in a significant rise of the melting temperature by 1.4 +/- 0.3 degrees C after 24 h (P less than 0.05) and 5.5 +/- 0.3 degrees C after 96 h (P less than 0.0001). G4 alone had no effect. Exposure of NH to NiAcet + G4, but not to the individual chemicals, slowly decreased solubility of the histone components in 0.2 M H2SO4. Only trace amounts of histones could be extracted from NH with acid after 72-h exposure to NiAcet + G4. Treatment of free histones with NiAcet, Ni3S2 and/or G4 resulted in a slow random polymerization of the proteins by NiAcet + G4, Ni3S2 + G4 and Ni3S2 alone, but not NiAcet or G4 alone. The action of Ni3S2 alone was slower than that of either nickel compound combined with G4. The present findings indicate that nickel carcinogens NiAcet and Ni3S2, in the presence of G4 or even alone (Ni3S2), are capable of causing protein-protein and perhaps also protein-DNA crosslinking. Reactions of this type may be involved in the mechanism(s) of nickel carcinogenesis.

Cadmium carcinogenesis in male Wistar [Crl:(WI)BR] rats: dose-response analysis of tumor induction in the prostate and testes and at the injection site.

Carcinogenic dose-response effects of CdCl2 in male Wistar [Crl:(WI)BR] rats were studied over a 2-year period. Groups of rats received a single s.c. injection of CdCl2 at doses of 0, 1.0, 2.5, 5.0, 10.0, 20.0, or 40.0 mumol/kg in the dorsal thoracic midline. Other groups received either four separate s.c. doses of 5 mumol Cd/kg each (at 0, 48, 96, and 168 h), or low dose cadmium (5.0 mumol/kg, s.c., at 0 h) followed by a higher dose (10.0 or 20.0 mumol/kg, s.c., at 48 h). The cadmium treatments resulted in appearance of tumors at the injection site, in the testes, and in the ventral prostate. Injection site tumors (mostly sarcomas) appeared to be strictly related to accumulated dose of cadmium and approached a 45% incidence at the highest cadmium dose (40 mumol/kg). Testicular tumors (mostly Leydig cell adenomas) were found to be highly dependent on testicular degeneration caused by cadmium. The highest Leydig cell tumor incidence occurred in the 40 mumol/kg (83%) and 20 mumol/kg (72%) dosage groups. Low dose pretreatment (5.0 mumol/kg) reduced or prevented the testicular degeneration and tumor formation that would otherwise result from a subsequent higher dose of CdCl2 (20 mumol/kg). Prostatic tumors (mostly adenomas of the ventral lobe) were also found to be associated with cadmium treatment, but in a non-dose related fashion. Prostatic tumor incidence was significantly elevated at the 2.5 mumol/kg dose of CdCl2 (eight tumors/26 rats; 31%) and showed a strong positive correlation between 0.0 and 2.5 mumol/kg in both tumor incidence and multiplicity. At higher doses, including those that caused marked testicular degeneration and induced prostatic atrophy, an elevated incidence of tumors did not occur. The occurrence of hyperplastic foci of the prostate, however, showed a strong positive correlation with increasing dose after single injections of cadmium up to and including 20.0 mumol/kg. Results indicate that CdCl2 can induce preneoplastic lesions of the prostate that appear to develop into tumors only at doses well below those causing marked degeneration of the testes and atrophy of the prostate.

Increased metallothionein gene expression in 5-aza-2'-deoxycytidine-induced resistance to cadmium cytotoxicity.

The pyrimidine analog, 5-azacytidine (AZA-CR), has been shown to increase the expression of the metallothionein (MT) gene and to induce tolerance to cadmium toxicity. Since incorporation into DNA of AZA-CR appears to be required for this effect, the deoxynucleoside of AZA-CR should also be effective. Therefore, this study was undertaken to assess the effect of 5-aza-2'-deoxycytidine (AZA-CdR) pretreatment on cadmium-induced cytotoxicity and MT expression in cultured cells. TRL 1215 cells in log phase of growth were exposed to AZA-CdR (0.4, 0.8, 4.0, 8.0 microM) followed 48 h later by the addition of cadmium (10 microM). MT concentrations were measured 24 h after the addition of cadmium. AZA-CdR alone caused modest, dose-related increases in MT levels (2.3-fold maximum), while cadmium alone resulted in a 9.5-fold increase. Pretreatment with AZA-CdR in combination with cadmium caused a 19--24-fold increase in cellular MT at all doses of AZA-CdR. Addition of the DNA synthesis inhibitor, hydroxyurea (HU), to the incubation medium during AZA-CdR exposure prevented the enhancing effect of the analog on cadmium induction of MT accumulation. Time course studies revealed that AZA-CdR pretreatment reduced the time required for cadmium to induce MT levels from 4--8 h to 0--2 h. AZA-CdR pretreated cells placed in suspension with cadmium (125 microM) showed a marked reduction in cadmium-induced cytotoxicity as reflected by reduced glutamic-oxaloacetic transaminase (GOT) loss. Uptake studies showed that AZA-CdR pretreatment had no effect on cadmium transport during the initial phases of exposure, indicating that an alteration in the toxicokinetics of the metal did not account for the reduction in toxicity. AZA-CdR did, however, cause hypomethylation of the MT-I gene. These results suggest that AZA-CdR pretreatment induces tolerance to cadmium toxicity by increasing the genetic expression of MT possibly through hypomethylation of the MT gene.

Induction of rat sarcomas in rats treated with antithymocyte sera after transplantation of human cancer cells.

Human cancer cells that had had high (greater than 160) tissue culture passages, when transplanted into antithymocyte-treated F344 newborn rats, caused induction of rat sarcomas in the rats within 2 or 3 subcultures, whereas human cancer cells with low (5-33) passages in vitro did not cause overt induction of rat sarcomas until after 5-10 subtransplantations. Because oncornavirus activity was not detected in either rat or human tumors, it is suggested that transforming sequences located on the human tumor cells may have been transferred to supporting rat reticulum cells in close contact with the human cancer cells.

Prevention of spontaneous leukemia in AKR mice by passive immunization and type specificity of this protection.

AKR mice were given sc injections of goat antiviral immune globulin from birth through 14 or 31 days of age. Although the shorter immunization schedule greatly reduced expression of spontaneous leukemia, the longer immunization period was required for essentially complete prevention of leukemia. Immune globulin with a high neutralization titer for ecotropic virus provided this protection, whereas antibody with a high neutralization titer for murine xenotropic virus did not.

Prevention of spontaneous leukemia in mice by oncornavirus immunization.

C57L, NIH, and SWR mice were immunized with inactivated Gross leukemia virus (GLV) and then mated with AKR males. Their F1 offspring were then immunized with the murine sarcoma virus pseudotype of GLV, MSV(GLV). The concentrations of infectious ecotropic AKR virus in tail extracts of immunized mice were 100- to 1,000-fold lower than in non-immunized controls when tested at 30--40 days of age. Although viral titers increased slightly with time, the titers remained at least one log10 lower in the immunized mice than in non-immunized F1 control mice at all times tested. The reduction in the level of expression of endogenous ecotropic virus showed a highly significant positive correlation with the reduction in incidence of spontaneous leukemia in these mice. These data thus show that successful immunoprevention of leukemia in mice can be achieved with viral vaccines.