Toxicological interactions between nickel and radiation on chromosome damage and repair.

Carcinogenic nickel compounds are usually found to be weak mutagens; therefore these compounds may not exert their carcinogenic activity through conventional genotoxic mechanisms. On the other hand, the activities of many nickel compounds have not been adequately investigated. We evaluated the genotoxic activities of nickel acetate using conventional chromosome aberration and sister chromatid exchange assays and found that there was no increase of chromosome aberrations or sister chromatid exchanges, although the highest dose (1000 microM) caused mitotic inhibition. In addition, we investigated its effect on DNA repair using our challenge assay. In this assay, lymphocytes were exposed to 0.1 to 100 microM nickel acetate for 1 hr during the G0 phase of the cell cycle. The cells were washed free of the chemical and, 1.5 hr later, were irradiated with two doses of gamma-rays (75 cGy per dose separated by 60 min). A significant dose-dependent increase of chromosome translocations was observed (p < 0.05). The increase is more than expected based on additive effects from exposure to nickel or gamma-rays individually. In contrast to the increase of chromosome translocations, there was no increase in chromosome deletions, although there was a nickel dose-dependent reduction of mitotic indices. Our data suggest that pretreatment with nickel interferes with the repair of radiation-induced DNA damage and potentially cause mistakes in DNA repair. Furthermore, we suggest that nickel-induced abnormal DNA repair may be a mechanism for its carcinogenic properties. The DNA repair problems that we observed after exposure to low doses of nickel may be viewed as a type of adaptive response.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
It is well recognized by scientists and by knowledgable lay people that many of our long-term health problems are due to chronic exposure to low doses of varying hazardous agents, including cigarette smoke and occupational exposures to known carcinogens (1,2). These exposures may induce detectable genetic alterations in the exposed populations, e.g., chromosome aberrations (3), which are indicative of long-term consequences. On the other This article was presented at the IV European ISSX Meeting on Toxicological Evaluation of Chemical interactions: Relevance of Social, Environmental, and Occupational Factors held 3-6 July 1992 in Bologna, Italy.
This study is partially supported by a grant from NIH (ROl ES 04926-02) and a contract from the Texas Legislature and the Texas Department of Health (IAC 1461). Dr. Heo was supported by a fellowship from Kangweon National University, Korea. The work of Miss Chiewchanwit is partially sup-hand, the exposure may not cause detectable genetic damage although the exposed populations may be at risk for adverse health outcomes. The latter phenomenon requires special attention and requires studies to elucidate further the mechanisms for induction of health effects. One such mechanism is the induction of DNA repair problems. Defects in DNA repair may cause cells to make serious mistakes from repairing innocuous DNA lesions to generating multiple genetic damage, such as chromosome instability. Such DNA repair errors allow genetic alterations relevant to the development of cancer to evolve. Therefore, defects in DNA repair can cause genetic instability and subsequently health effects. Carcinogenesis is a complicated process involving multiple and, often, sequential genetic alterations (4,5) so that errors in DNA repair may lead ultimately to carcinogenesis. Using an in vivo/in vitro mouse mammary tumor model, we have documented that the following sequence of events occurred before the cells became tumorigenic: chromosome instability, evolution of cells with stabilized but altered karyotypes, inactivation of the RB tumor suppressor gene and amplification of the cmyc oncogene (6)(7)(8). We hypothesize that the induced chromosome instability is due to induction of DNA repair problems and we have recently developed a challenge assay to detect this phenomenon (9). In this report, the activity of a nongenotoxic carcinogen, nickel, is presented.
Workers exposed to nickel compounds have a significantly increased risk for the development of lung cancers; therefore, these compounds are classified as human carcinogens (10)(11)(12). The properties of some of these compounds have been studied extensively and these findings have been summarized in several recent reviews (13)(14)(15). Among the different kinds of nickel compounds, insoluble compounds are usually more carcinogenic than the soluble ones. Although their carcinogenic properties are well documented, the mechanisms for these activities are not established yet. Due to the problems of establishing dosimetry for insoluble compounds, many of the genotoxic studies have been conducted with soluble ones. These reports indicate that most of the carcinogenic nickel compounds are inactive or weakly mutagenic in standard genotoxic assays.
Biochemical studies have documented that nickel can bind to DNA and protein in cells in vitro (16,17) and to chromatin in vivo (18). Such binding to cellular macromolecules is correlated with the compounds' ability to interfere with DNA syn-Environmental Health Perspectives thesis (16,19). Nickel compounds can induce slight increases in chromosome aberrations (16,20) and sister chromatid exchanges (21,22), and tend to be weak mutagens (21,23).
Since nickel compounds are not strong mutagens by themselves, some investigators feel that they may be potent co-mutagens. In fact, nickel compounds can enhance the induction of mutations by methylmethane sulfonate in bacteria (24). In mammalian cells, they can enhance the induction of mutation by ultraviolet light (21), single stranded DNA breaks by X-rays, and cell transformation by benzo[a]pyrene (25). This enhancement activity of nickel compounds may be due to their effect on DNA repair. On the other hand, no enhancement effect was detected in a CHO/ HGPRT gene mutation assay (26). Nevertheless, the mechanisms for the potential synergistic effects need to be elucidated.
We have systematically investigated the cytogenetic effects of a soluble nickel compound, nickel acetate. In addition, we have used our challenge assay to elucidate cytogenetically its effects on DNA repair. We have chosen to study nickel acetate because it is a soluble carcinogenic compound (27)(28)(29)(30) and its clastogenic effects have not been adequately investigated.
The challenge assay is based on our hypothesis that chemicals that can bind to cellular macromolecules are able to interfere with normal cellular functions such as DNA repair processes and can cause mistakes in DNA repair (9). The assay is conducted by exposing cells first to the target chemical (pulse-treatment) and then to ionizing radiation. These cells are therefore challenged to repair the radiation-induced DNA lesions after chemical exposure so that repair occurs in the absence of the chemical. If pretreatment with the target chemical causes errors in the repair process, mistakes in repair would occur. One type of such mistakes is the rejoining of DNA fragments to the wrong DNA molecules which leads to the formation of rearranged chromosomes. Since radiation-induced DNA damage is repaired within a very short time (during the GO phase of the cell cycle in our protocol), the observed abnormal chromosomes are, therefore, not caused by replication errors. Thus, we interpret our assay data to indicate DNA repair problems.
The feasibility of the challenge assay has been tested with lymphocytes from cigarette smokers. We observed that cells from smokers have significantly more dicentric chromosomes than those from nonsmokers after the challenge with X-rays (31). The most effective protocol for detecting the difference was found to be the exposure of lymphocytes to a double dose of 100 cGy of X-rays each separated by 60 min. Therefore, the double exposure protocol was used in this study. Furthermore, the exposure doses were reduced from 100 cGy to 75 cGy per dose since the detection of effects using low doses is more relevant than those with higher doses. In this study, we found that nickel acetate does not cause chromosome aberrations or sister chromatid exchanges in human lymphocytes even when cytotoxic doses are used. On the other hand, this compound does induce abnormal DNA repair.

Materials and Methods Source of Lymphocytes and Blood
Culture Procedures Blood samples are obtained from normal volunteers who are healthy and not occupationally exposed to known hazardous agents. Most of the experiments are conducted with blood samples from two male donors. The samples are collected by venipuncture in the presence of sodium heparin as anticoagulant. After collection of samples, whole blood cultures are set up using phytohemagglutinin as mitogen and RPMI 1640 culture medium according to the techniques established in our laboratory (31). The only exception is that autologous plasma from each donor is added to each culture to enhance cell growth (0.25 ml per 10 ml culture).

Chemicals
Nickel acetate is purchased from Alfa Products (Danvers, MA). An appropriate amount of the compound is weighed out for use in each experiment. The compound is dissolved in distilled water, filter sterilized and diluted with sterile distilled water to the desired concentrations.

Irradiation Conditions
Blood cultures are irradiated in a Mark I Cesium-137 Pneumatic Irradiator which is located in the Department of Radiation Therapy. The dose rate is set at 80 cGy/min. For irradiation, tubes containing blood cultures are loaded onto a styrofoam-type holder which can receive six tubes for irradiation at one time. The holder is placed in the center of the cavity of the irradiator and on top of a rotating platform turning with a speed of approximately 10 revolutions/min.

Treatment Conditions
Cells are either treated during the GO or the Gl/S phases of the cell cycle. For treat-ment during the GO phase, cells are treated with chemicals and/or radiation before cultures are set up. For treatment at the G 1/S phases, cells are treated at 24 hr after initiation of cell cultures. Each culture is treated with 0.1 ml of the appropriate concentrations of nickel acetate.
Chromosome Aberrations and Sister Chromatid Exchanges. For treatment at the GO phase of the cell cycle, cells are treated with chemicals for 1 hr, washed free of the chemicals (twice with excess amount of Hank's Ca++ and Mg++ free balanced salt solution), and then cultures are initiated. At 16 to 24 hr after initiation, bromodeoxyuridine (BrdU) is added to each culture to achieve a final concentration of 5 pM. At 52 hr after initiation of cultures, cells are harvested, stained with fluorescentplus-Giemsa technique and analyzed for the presence of chromosome aberrations according to our standardized technique (31). Two experiments are performed and 100 cells per experiment are analyzed for the presence of chromosome aberrations.
For treatment at the G1/S phases of the cell cycle, cells are exposed to different concentrations of the chemical from 28 hr until harvest time. BrdU is added to cultures at the same time. At 52 hr after initiation of cell cultures, cells are harvested for chromosome aberration and at 72 hr for sister chromatid exchange analyses. Three experiments are performed and 50 cells per experiment are analyzed for chromosome aberrations and for sister chromatid exchanges.
Challenge Assay for DNA Repair. Cells are treated with different concentrations of the chemical at the GO phase of the cell cycle for 1 hr and washed free of the chemical as described earlier. At 1.5 hr after termination of treatment with chemical, cells are irradiated with two doses of y-rays (75 cGy per dose and separated by 60 min). The radiation doses for this experiment are less than those of our earlier experiment (31 ) because our emphasis is to identify biological responses using as reduced doses of chemical and radiation as possible. Furthermore, y-rays instead of Xrays are used because the former machine is more readily available for us to conduct studies. Furthermore, the biological effects of these two kinds of radiation are very similar. Cell cultures are initiated after the completion of irradiation. At 52 hr after initiation, cells are harvested for chromosome aberration analyses. Four   NA 0 Ml 0 Ml Abbreviations: NA, not analyzed; Ml, mitotic inhibition. a Lymphocytes were exposed to the chemical for 1 hr before initiation of culture (GO) or from 28 hr until harvest time (Gl/S). Chromosome-type abnormalities are rarely observed. a Lymphocytes were exposed to the chemical for 1 hr at GO, washed and irradiated 1.5 hr later with two doses of y-rays separated by 60 min. Cultures were initiated after the treatment. Our challenge assay is used to investigate whether exposure to nickel acetate can cause mistakes in DNA repair and lead to the formation of abnormal chromosomes. The frequencies of dicentric chromosomes obtained from four independently conducted studies are summarized in Table 2. As shown in the table, the collected data are rather consistent from one experiment to another. Additional data from these four experiments are summarized in Table 3. As shown in the table, the chemical induces a significant dose-dependent increase of chromosome-type rearrangements (p<0.05) as determined by the analysis of variance procedure although the response falls off at high doses of nickel acetate (100 pM). In addition, the translocation frequencies in cells treated with 0.1, 1.0, and 10 pM nickel acetate are significantly different from each other (p<0.05; contrast analysis). The frequencies for those cells treated with 10 pM of the chemical is significantly different from the control (p<0.05; contrast analysis). The observed phenomenon is induced by nickel acetate with concentrations that are not clastogenic by themselves. On the other hand, no change in chromosome-type deletions is observed. In addition, there is a nickel dose-dependent reduction of mitotic indices.

Discussion
Carcinogenic nickel compounds are found to be weakly mutagenic in a variety of shortterm assays, therefore, these compounds may not exert their carcinogenic activities using conventional genotoxic mechanisms.
Volume 102, Supplement 9, November 1994 A unique observation is that these compounds appear to affect heterochromatin preferentially. Nickel-transformed Chinese hamster embryo cells have nonrandom deletion of the heterochromatin in the Xchromosome (32). On the other hand, other investigators have shown that nickel compounds can enhance the activities of mutagenic agents (21,24,25). However, the mechanisms for expression of these phenomena have not yet been elucidated.
Since nickel compounds are capable of binding to cellular DNA and proteins (16)(17)(18), they can cause abnormal expression of cellular functions. We hypothesize that one of those functions is fidelity of DNA repair. Using a challenge assay, we have shown that noncytotoxic and nonclastogenic doses of nickel acetate induce significant increase of rearrangement-type chromosome aberrations (p<0.05; Table 3). On the other hand, the frequency of deletion-type chromosome aberrations is unchanged after exposure to different combinations of nickel and y-rays. The latter observation suggests that the amount of DNA strand breaks induced by y-rays may not have been increased significantly by prior exposure to different concentrations of nickel. The increased frequency of rearranged chromosomes is therefore caused by problems in repair of radiationinduced DNA damage. One of the causes for the problem is the inability to rejoin radiation-induced DNA fragments so as to reform the original DNA molecules. This can be caused by interference from complexes formed between nickel and DNA and between nickel and repair enzymes. Another possibility is due to nickelinduced delay of DNA repair. Delay in repair may allow a significant amount of damage induced by the first dose of radiation to interact.with damage induced by the second dose of radiation (60 min later). The phenomenon is well documented in experiments which showed that inhibition of DNA repair by ara-C after exposure to X-rays causes significant increase of rearrangement-type chromosome aberrations (33,34). Both mechanisms may exist for nickel under our experimental conditions. Therefore, we interpret our data to be indicative of infidelity of DNA repair caused by exposure to nickel.
A wide range of concentration of nickel acetate (from noncytotoxic to cytotoxic doses) is tested in our assay. The shape of the dose-response curve (last two columns of Table 3) reflects the biological effects from exposure to these different doses of nickel. Although we have not conducted mechanistic studies to elucidate the cause of the response, there are data from the literature to explain our observed phenomenon. It is possible that nickel induces an adaptive cellular response (35). The study showes that after exposure of cells to a low dose of a DNA damaging agent, cells become resistant to damage from exposure to a high dose of another agent. Induction of DNA repair enzymes is a possible explanation for this phenomenon. Our data indicate that the induced adaptive response may not always be beneficial.
Exposure to the highest dose of nickel leads to a translocation frequency lower than that from the second highest dose. This response may be due to toxicity of nickel to lymphocytes. It may, on the other hand, be due to reduced intranuclear concentration of nickel at this extracellular dose. For example, in an in vivo study, the tissue concentrations of nickel increase with time, as expected, after exposure to increasing doses of nickel carbonate (18). However, at certain tissue concentrations of nickel, the concentrations in cell nuclei are actually reduced. This study suggests that the nucleus has specific mechanisms to remove nickel after certain extracellular concentrations of nickel are reached. The same mechanism may exist in lymphocytes in our study.
Infidelity of DNA repair is probably an important causal mechanism in the development of cancer. Mistakes in the repair of spontaneous or induced DNA lesions may convert generally innocuous DNA damage into a major defect that can cause longterm health effects. It is conceivable that infidelity of DNA repair creates instability of the genome which permits genetic alterations significant in the development of cancer to occur. In fact, the observation of altered p53 genes in cells exposed to nongenotoxic nickel is consistent with our suggested activity of nickel (36). The well documented increase of cancer risk among patients with DNA repair defects and/or chromosome instability syndromes is also consistent with the hypothesis. Therefore, infidelity of DNA repair may contribute to the production of multiple and sequential genetic alterations within the same cell for clonal adaptation and progression of abnormal cells. This is the hallmark for development of cancer (4,5,37,38). Our data suggest that nickel acetate, and perhaps other carcinogenic nickel compounds, can cause DNA repair problems and that this activity may be one of the mechanisms responsible for their carcinogenic properties. Our suggestion is also consistent with the observation of chromosome abnormalities in mouse tumors induced by nickel compounds which are not clastogenic (39).
Our challenge assay is designed with the intention of using it in population monitoring studies. Therefore, cells are treated with chemicals during the GO phase of the cell cycle in order to mimic in vivo exposure to chemicals. As part of our in vitro assay, cells are then exposed to radiation in the absence of chemicals. As demonstrated by our study with lymphocytes from cigarette smokers (31), these lymphocytes are exposed to cigarette smoke in vivo and then to radiation in vitro. In that study, our data indicate that exposure to cigarette smoke causes DNA repair problems. After the challenge assay is better characterized, it may be used to detect DNA repair problems in cells from workers who are occupationally exposed to potentially hazardous agents.