Metallothionein-I/II Double Knockout Mice Are No More Sensitive to the Carcinogenic Effects of Nickel Subsulfide than Wild-Type Mice

Abstract

Metallothionein (MT) is a high-affinity metal-binding protein thought to mitigate the toxicity of various metals. MT may limit the toxicity of a metal by direct binding or through action as an antioxidant for metals that generate reactive oxygen species. Nickel compounds have carcinogenic potential in humans and animals, possibly by production of oxidative stress. The impact of MT deficiency on the carcinogenic effects of nickel is unknown. Thus, groups (n = 25) of male MT-I/II double knockout (MT-null) or MT wild-type (WT) mice were exposed to a single treatment of nickel (0.5 or 1.0 mg Ni₃S₂/site, intramuscularly, [i.m.], into both hind legs), or left untreated (control) and observed over the next 104 weeks. There were no differences in the incidence of spontaneous tumors in MT-null and WT mice. Nickel induced injection site fibrosarcomas in a dose-related fashion to a similar extent in both WT and MT-null mice. Nickel-treatment had no effect on total lung tumor incidence, although some phenotypic-specific differences occurred in the proportion of benign and malignant pulmonary tumors. Overall, MT-null mice appear no more sensitive to the carcinogenic effects of nickel than WT mice. Thus, poor MT production does not appear to be a predisposing factor for nickel carcinogenesis.

Metallic agents are an important class of carcinogens. They include the transition metal nickel, which is considered a human carcinogen. Occupational exposures to nickel in humans are associated with development of cancers, and the metal is an effective rodent carcinogen (for review see IARC 1990; Kasprzak, Sunderman, and Salnikow 2003). Carcinogenic nickel compounds target the respiratory tract, producing nasal and pulmonary malignancies after inhalation in humans, while in rodents inhalation of certain nickel compounds produces lung cancers and systemic nickel exposure can induce pituitary or pulmonary tumors. Nickel can also induce local malignancies, which are primarily sarcomas, in rodents at the site of repository-type injections.

Metallothionein (MT) is a low-molecular-weight, metal-binding protein, which is distinctive in its numerous cysteines and lack of aromatic amino acids (Klaassen, Liu, and Choudhuri 1999; Davis and Cousins 2000; Waalkes and Perez-Olle 2000; Sato and Kondoh 2002). MT is a key part of the biological response to toxicological stress induced by a variety of metals (Klaassen, Liu, and Choudhuri 1999; Waalkes and Perez-Olle 2000; Sato and Kondoh 2002). MT synthesis is highly inducible and is markedly elevated in response to exposure to many toxic metals (Waalkes and Perez-Olle 2000). Because MT will avidly bind many metals, sequestration is likely to be one way in which this protein detoxicates metals (Klaassen, Liu, and Choudhuri 1999; Waalkes and Perez-Olle 2000; Sato and Kondoh 2002). MT also appears to be able to scavenge oxygen free radicals and thereby reduce oxidative tissue damage from physically or chemically induced oxidative stress (Sato and Kondoh 2002). This includes oxidative stress induced by certain metals (Sato and Kondoh 2002). Indeed, MT-deficient cells are hypersensitive to oxidant injury (Lazo et al. 1995; Suzuki, Apostolova, and Cherian 2000), and it is suspected that MT may play a role in regulation of intracellular redox status (Lazo et al. 1995). In this regard, it is thought that generation of reactive oxygen species (ROS) may be a critical factor in nickel-induced genotoxicity and, consequently, carcinogenesis (Kasprzak 1995; Kasprzak, Sunderman, and Salnikow 2003; Costa et al. 2002). There is evidence that MT is associated with nickel exposure and can alter nickel toxicity (Maitani and Suzuki 1983; Sunderman and Fraser 1983; Bracken and Klaassen 1987; Srivastava et al. 1993; Cheng et al. 2003). For instance, nickel treatment will induce MT synthesis both in vivo and in vitro (Maitani and Suzuki 1983; Sunderman and Fraser 1983; Bracken and Klaassen 1987; Srivastava et al. 1993; Cheng et al. 2003). Furthermore, increased MT expression in mice has been associated with a reduction in acute toxicity and tissue oxidant injury resulting from the nickel exposure (Srivastava et al. 1993). However, a recent study in mice with genetically engineered overexpression of MT found that nickel induced injection site sarcomas at a rate similar to MT wild-type (WT) mice that expressed MT at normal levels (Waalkes et al. 2004). The impact of MT deficiency on nickel carcinogenesis has not been studied.

Although there is evidence that MT can perturb some of the toxic effects of nickel, possibly including nickel-induced oxidant injury, what impact MT deficiency has on the carcinogenicity of nickel is unknown. Thus, this study sought to test the hypothesis that poor expression of MT might enhance the carcinogenic effects of nickel using genetically engineered MT-null mice. Overall, the results indicate that MT-null mice are no more sensitive to the carcinogenic effects of nickel than WT mice.

MATERIALS AND METHODS

Chemicals

Nickel subsulfide (Ni₃S₂; > 99.9% pure) containing smaller than 30 μm particles was obtained from INCO Ltd. (Toronto, Canada). Glycerol was obtained from Sigma Chemical Co. (St. Louis, MO).

Animals and Treatments

Animal care was provided in accordance with the U.S. Public Health Policy on the Care and Use of Animals as defined in the Guide to the Care and Use of Animals (NIH Publication 86-23). Homozygous MT-I/II knock-out mice (129S7/SvEvBrd-Mt1 ^{tm 1/ Bri} Mt2 ^{tm 1/ Bri} /J) (Masters et al. 1994) and corresponding WT (129S3/SvImJ) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). The homozygous mutants were mated inter se to maintain the line. Mice were housed in a standard barrier facility at a temperature of 68°F to 72°F, with a relative humidity of 50% ± 5%, and a 12-h light/dark cycle. A basal diet (NIH-31 Block Modified 6% fat; Ralston Purina, St. Louis, MO) and acidified water were provided ad libitum. The National Cancer Institute, Frederick, animal facility, where the biopsy portion of the present study was conducted, and its animal program are accredited by the American Association for Accreditation of Laboratory Animal Care.

A total of 75 WT and 75 MT-null male mice were randomly divided into three groups per phenotype of 25 each. At 12 weeks of age separate groups of MT-null and WT animals received a one time nickel exposure consisting of intramuscular (i.m.) injections of Ni₃S₂ suspended in 50% aqueous glycerol into both thighs at doses 0.5 or 1.0 mg Ni₃S₂/site (0.05 ml/site). Controls were untreated. The point of injection was defined as time = 0. The mice were then observed for an additional 104 weeks. The doses of nickel were selected on the basis of prior work showing that they induce tumors in mice (Waalkes et al. 2004). This study was performed contemporaneously with a study on cisplatin carcinogenesis in MT-null and WT mice that jointly used the same untreated MT-null and WT control animals as in the present study. The results of this study with cisplatin are reported elsewhere (Waalkes et al. 2005).

Clinical Data and Pathology

Individual body weights were recorded at intervals of every 5 weeks. Clinical signs were checked daily. Mice were killed with CO₂ when moribund or at the study termination. A complete necropsy was performed on all moribund animals, animals found dead, or at the terminal sacrifice. The following tissues were taken and processed for histological analysis: injection sites, testes, liver, kidneys, lung, adrenal, spleen, thymus, pituitary, and all grossly abnormal tissues. Tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin for histological analysis. All pathological assessments were performed in a blind fashion.

Data Analysis

Data are given as incidence or as mean ± SEM, as appropriate. A probability level of p ≤ .05 was considered to indicate a significant difference in all cases. In defining the incidence of mice bearing benign or malignant tumors in cases of multiple tumors in the same tissue, the animal was assigned to the appropriate group based on its most advanced lesion. In pairwise comparison of tumor incidence and survival, a two-sided Fisher’s exact test was used. For multiple comparisons of mean body weight data a two-sided Dunnett’s test was used. Tumor incidence is based on numbers of animals available for pathological analysis, and loss of animals to observation was typically due to autolysis that was considered too advanced for appropriate diagnosis. A total of five mice were lost to observation, including two in the group of MT-null mice treated with the higher dose of nickel and one each in the following groups: WT control, MT-null control, MT-null low-dose nickel, MT-null high-dose nickel. All other animals were considered to be at risk for tumor development.

RESULTS

Survival

Groups (n = 25) of male MT-null and WT mice received a single treatment of nickel (0.5 or 1.0 mg Ni₃S₂/site in both thighs) whereas controls (n = 25) of both phenotypes were left untreated. Mice were killed when moribund or after 104 weeks. Survival at selected times during the study is shown in Table 1. There were no differences in survival between control MT-null mice and control WT mice at any time during the study. The doses of nickel used were not overtly toxic and no mice died during the first week after nickel injection; only two nickel-treated mice died during the first 35 weeks. Nickel exposure did reduce survival in both WT and MT-null mice compared to respective controls at later time points. Reduced survival after nickel treatment started occurring at between 50 and 60 experimental weeks in both MT-null and WT mice, which coincides with the appearance of nickel-induced injection site sarcomas (see below). In nickel-treated groups, no significant differences in survival were observed between the two phenotypes (WT and MT-null) at any age.

Body Weights

There were no differences in body weight between control WT and control MT-null mice at any point in the study (Figure 1). However, nickel-treated MT-null mice showed significantly lower body weights (up to 20%) at the high dose (1.0 mg Ni₃S₂/site) compared to phenotype-matched control from experimental week 60 until the end of the study and at the low dose (0.5 mg Ni₃S₂/site) from week 70 on. WT mice treated with the higher dose of nickel showed reduced body weights (maximum 13%) compared to control from experimental week 50 on, but WT mice treated with the lower dose of nickel did not show significantly lower body weights. In nickel treated groups within a phenotype, there were no significant differences in body weight between the two dosage groups (0.5 or 1.0 mg Ni₃S₂/site) at any age. Similarly, no significant differences in body weights occurred between dosage-matched MT-null and WT mice at any age.

Injection Site Tumors

Nickel exposure induced malignant injection site tumors in high incidence regardless of mouse phenotype (Table 2). Clear, dose-related increases in injection site fibrosarcomas occurred in both WT and MT-null mice. The maximum incidence was 72% in WT mice and 65% in MT-null mice given the higher dose of nickel. There were three instances in which animals had bilateral fibrosarcomas; two in the WT mice treated with the higher nickel dose and one in a high dose MT-null mouse. One injection site fibrosarcoma in a MT-null mouse given the higher dose of nickel metastasized to the lung. The first nickel-induced injection site tumor occurred at 40 weeks.

Lung Tumors

Pulmonary tumors were a common occurrence in control mice of both phenotypes and nickel treatment did not impact total lung tumor incidence (Table 3). However, nickel treatment did have mixed effects on the relative proportion of benign adenoma and malignant adenocarcinoma of the lung. In WT mice the lower dose of nickel caused a significant increase in pulmonary adenocarcinoma incidence, but not in adenoma or total tumor incidence, when compared to WT control. In contrast, nickel injection at the lower dose significantly increased lung adenoma incidence in MT-null mice compared to dosage-matched WT mice but not compared to MT-null control mice. In contrast, the higher nickel dose significantly reduced lung adenoma incidence in MT-null mice compared to phenotypic control or dosage-matched WT mice.

Spontaneous Tumors

Various spontaneous tumors occurred in this study. These included six hepatocellular adenoma, two testicular teratoma, and a pituitary adenoma in WT control. Three hepatocellular adenoma, two liver hemangioma, one hepatocellular carcinoma, and two testicular teratoma occurred in WT mice treated with 0.5 mg Ni₃S₂/site. One hepatocellular adenoma and one hepatocellular carcinoma occurred in WT mice treated with 1.0 mg Ni₃S₂/site. One nasal turbinal osteoma and two hepatocellular adenoma occurred in control MT-null mice. In MT-null mice treated with 0.5 mg Ni₃S₂/site a lymphoma, an adrenal sarcoma, a hepatocellular adenoma, two hepatocellular carcinoma, and an intestinal adenoma were observed while a pancreatic carcinoma and a hepatocellular carcinoma occurred in a MT-null mouse given 1.0 mg Ni₃S₂/site.

DISCUSSION

Our initial hypothesis was that MT might mitigate nickel carcinogenesis and that, therefore, MT deficiency would enhance tumor formation after nickel exposure. However, the results of the present study clearly show that the inability to express the major forms of MT had no impact on the carcinogenic effects of nickel. In this regard, nickel induced injection site fibrosarcomas at essentially equal incidence in both phenotypes. The absence of a significant impact on nickel carcinogenesis in the present work is consistent with prior work showing MT-transgenic mice, which overexpress the protein at levels up to 20-fold more than WT controls (Iszard et al. 1995), were no less sensitive to nickel carcinogenesis (Waalkes et al. 2004). Indeed, MT-transgenic mice showed a similar incidence of nickel-induced injection site fibrosarcomas compared to WT mice (Waalkes et al. 2004) after injection of Ni₃S₂ at the same doses used in the present study. MT-null mice are deficient in MT in all major MT-producing tissues (Liu et al. 1996) and recent data in genetically unaltered mice indicate that skeletal muscle has significant basal levels of MT that can be increased by a variety of treatments (Jahgoe et al. 2002; Rubenstrunk et al. 2003; Lecker et al. 2004). MT does not avidly bind nickel (Waalkes, Harvey, and Klaassen, 1984), although the protein appears to be able to reduce ROS toxicity (Sato and Kondoh 2002). Nickel may well induce ROS as part of its carcinogenic mechanism (Kasprzak 1995; Kasprzak, Sunderman, and Salnikow 2003; Costa et al. 2002). However, from the current results it appears that any impact MT might have in reducing nickel related ROS-induced damage has limited impact on eventual carcinogenesis. In fact, several studies concerning acquired nickel tolerance at the cellular level indicate that this tolerance is based on oxidative stress responses that have little to do with MT (Imbra, Wang, and Costa 1989; Salnikow et al. 1994). Although there is limited evidence that MT may mitigate some of the acute toxic effects of nickel (Srivastava et al. 1993), this protection does not appear to extend to nickel-induced events leading to carcinogenesis.

With regard to pulmonary tumors, the incidence of total lung tumors was not increased by the nickel injections in either phenotype when compared to control. Nickel treatment did have mixed effects on the relative proportion of benign and malignant lung tumors. In this regard, the lower dose of nickel induced an increase in adenocarcinomas in WT mice compared to control, which was not observed at the higher dose or in MT-null mice at either dose. In addition, MT-null mice had more lung adenoma at the low dose of nickel when compared to dosage-matched WT mice and less lung adenoma at the high dose of nickel when compared to dosage-matched WT and MT-null control mice. Because these events occurred in apparent isolation and showed no dose-response relationships, they should be interpreted with caution. The inconsistent nature of these results perhaps point towards chance events rather than events of biological significance. Similarly, McNeill, Chrisp, and Fisher (1990) found intraperitoneal injections of Ni₃S₂treatments did not produce evidence of dose-related increases in pulmonary tumors in strain A/J mice.

In summary, MT-null mice appeared no more sensitive to nickel carcinogenicity than mice expressing normal levels of MT. These results are consistent with data indicating that engineered overexpression of MT in mice does not mitigate the carcinogenic effects of nickel (Waalkes et al. 2004).