Molecular and Cellular Biochemistry 255: 3–10, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
3 Conference overview
Metal-induced toxicity, carcinogenesis, mechanisms and cellular responses Stephen S. Leonard,1 Jacquelyn J. Bower1,2 and Xianglin Shi1 1
Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV; 2Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV, USA
Abstract A wide variety of metals have been reported to act as mutagenic and carcinogenic agents in both human and animal studies. The underlying mechanisms are being extensively investigated. Recently, a new sub-discipline of molecular carcinogenesis has surfaced and new techniques and instruments are being developed which allow exploration of the complex biological relationships and signaling pathways involved in response to metal exposure at the molecular level. The 2nd Conference on Molecular Mechanisms of Metal Toxicity and Carcinogenesis was held at NIOSH in Morgantown, West Virginia, Sept. 8–11, 2002. One hundred thirty scientist from sixteen countries presented their novel findings and investigations of metal-induced carcinogenesis. The conference focused on state-of-the-art research and developments in metal toxicity and carcinogenesis. Emphasis was placed on delineating molecular mechanisms involved in free radical effects, cellular uptake, signaling pathways/interaction, dose response, biomarkers, and resistance mechanisms. This article reviews some of the novel information presented at the conference and discusses future avenues of research in this field. (Mol Cell Biochem 255: 3–10, 2004) Key words: metals, carcinogenesis, reactive oxygen species, transcription factors, NF-κB, AP-1 and MAP kinases
Introduction Exposure to certain metals in both environmental and occupational settings has been reported to be carcinogenic. Although, a wide variety of metals, including arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), nickel (Ni) and vanadium (V), have been indicated as carcinogenic agents in both animals and humans, the underlying mechanism(s) are not well understood. These metals may share a common pathway, but in some cases can diverge into distinctly different routes of cancer development. The metal involved, the dose, the duration of exposure, the location of exposure, and other environmental conditions may all contribute to determining the pathway of cellular responses leading to carcinogenesis. Animal and epidemiological studies have identified several metals and metal-containing compounds as potent mutagens and carcinogens [1–4]. Over the past two decades, chemical and cellular studies have contributed enormously to our un-
derstanding of metal-induced carcinogenesis. The process of carcinogenesis is considered to have four stages: initiation, promotion, progression, and metastasis [5–7]. Metals can cause genotoxicity and carcinogenicity through different cellular pathways. Metals can also enhance a biological effect by itself or with other factors in an additive or synergistic manner [8–10]. Although many metals have been shown to have a unique mechanism of action, several common pathways, including those initiated by oxidative stress, may be shared by several of these carcinogenic metals. While mutations of genomic DNA are traditionally considered to be important in the initiation phase of cancer development, activation of transcription factors and the amplification of oncogenes also contribute to the tumor initiation process [11–13]. The promotion and progression phases of cancer development can be affected by changes in gene expression and cellular signaling pathways [14–17]. Metalinduced carcinogenesis has been found to be involved in all phases of cancer development.
Address for offprints: S.S. Leonard, Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, WV 26505, USA (E-mail:
[email protected])
4 The development of new techniques to study the mechanisms of carcinogenesis has given us the ability to further define the pathways involved in this process. These new molecular techniques are generating a significant amount of novel information about metal-induced carcinogenesis in the following areas: (a) effects of reactive oxygen species (ROS) and redox signaling on cellular function; (b) regulation of nuclear transcription factors, such as NF-κB, AP-1, and NFAT; (c) activation of p53 tumor-suppressor genes and metal-induced expression of antioxidant-related genes; (d) metalinduced apoptosis; (e) effects of metals on cell growth regulation; and (f) metal-induced oncogene expression. Metals and metal compounds are found throughout cellular organelles and cellular components including membranes, mitochondria, endoplasmic reticulum, the nucleus, and lysosomes [18, 19]. Metals are involved in cellular functions such as production of energy in the electron transport chain, detoxification, enzymes, and enzyme cofactors [20, 21]. Metal ions can also bind directly to cellular components via ionic or coordinating bonds. This is one of the causes of site-specific damage, which can be done by metals [22, 23]. Metal can bind to both DNA and proteins causing lesions, strand breaks, as well as base modifications [24, 25]. This type of metal-induced damage is one of the ways which metal can affect cellular responses. Recent investigations are providing further information on the role of ROS on metal-induced carcinogenesis. The role ROS appears to be an important and nearly universally common step in metal-induced cellular responses [26–28]. These ROS include superoxide radical (O2.–), hydrogen peroxide (H2O2), hydroxyl radical (.OH), nitric oxide (NO.), peroxyl radical (ROO.), hypochloride (HOCl), alkoxyl radical (RO.), and thiyl radical (RS.). Some metal ions, such as Cr(VI), are capable of producing ROS in a cellular environment through a Fenton-like reaction or the Haber-Weiss cycle [29–31]. This cyclic reaction can cause chronic production of ROS at a specific location [32, 33]. The ROS generated in these systems have been shown to be responsible for metal-induced cellular injury [34, 35]. Recent studies have used new methods and technology to elucidate the pathways utilized in metal-induced carcinogenesis. The interrelationship and involvement of the signaling pathways and cellular responses are also being studied and further defined. The 2nd Conference on Molecular Mechanisms of Metal Toxicity and Carcinogenesis held at the National Institute for Occupational Safety and Health (NIOSH), Morgantown, WV, USA from Sept. 8–11, 2002 was a continuation of the first one, also held in Morgantown in 2000. One hundred thirty scientists from sixteen countries presented their novel investigations concerning metal-induced carcinogenesis, the role of ROS, signaling pathways, metal-induced cytotoxicity, the mechanisms involved in these processes, and therapeutic/preventative strategies. The conference focused
on state-of-the-art information concerning recent developments in metal toxicity and carcinogenesis. In addition to the molecular mechanisms of carcinogenesis mentioned above, the following areas were also emphasized during the meeting: oxidation and free radical effects; cellular uptake and dose-response; development of biomarkers; resistance mechanisms; reproductive toxicity; and risk assessment.
Reactive oxygen species (ROS) in metal exposure; generation and mechanisms of action Epidemiological, animal, and cellular studies have established that Cr(VI) compounds are toxic and carcinogenic [23, 36]. Occupational exposure to Cr(VI)-containing compounds is known to induce lung toxicity and increase incidence of cancers of the respiratory system [13, 18, 23, 37]. In experimental animals, Cr(VI) compounds have been shown to induce tumors at the injection and implantation sites [37–39]. For example, intra-bronchial implantation of chromate compounds in rats resulted in bronchial carcinomas, whereas intramuscular implantations and intra-pleural or subcutaneous injections in rats and mice result in injection site sarcomas [13, 38]. Cr(VI) have been shown to cause bacterial mutation and transformation in mammalian cells [12, 40]. Although the mechanism is still not well understood, it is generally considered that the carcinogenic effect of Cr(VI) involves cellular uptake because Cr(VI), not Cr(III), which actively enters cells by the sulfite transport mechanism [21]. Once inside the cells, Cr(VI) is reduced to its lower oxidation states, such as Cr(V) and Cr(IV). These reactive intermediates can directly cause DNA damage, are also capable of generating ·OH radical from H2O2 via a Fenton-like reaction [41]. During the Cr(VI) reduction process, molecular oxygen is consumed to generate O2·– radical and H2O2 [41– 43]. Thus, in the cellular system, Cr(VI) can generate a whole spectrum of ROS. Through ROS mediated reactions, Cr(VI) causes DNA damage [44], activation of nuclear transcription factors (NF-κB, AP-1, and p53) [15, 16, 45], apoptosis [46], cell growth arrest [47], oncogene expression [17], and activation of certain members of the mitogen-activated protein (MAP) kinase family [15]. Arsenic has been found to be carcinogenic in lung, liver, skin, bladder, and kidney. The mechanism of As carcinogenesis is largely unknown. Some mechanisms and modes of action have been proposed to include ROS production leading to oxidative stress, mutation, cell proliferation, and inhibition of DNA repair. Oxidative stress, induced by arsenic exposure, and its possible sources was discussed by Kitchen and Ahmad at the meeting. Arsenic induced oxidative stress might result from direct production of ROS, release of iron
5 from ferritin, redox cycling of different forms of As, or arsenic radical species. Carcinogenesis due to arsenic exposure is hypothesized to proceed via .OH production from the Fenton reaction, the Haber-Weiss reaction, or by other mechanisms which remain to be identified. Defining the mechanisms of arsenic-induced ROS generation and related cellular injuries is required to understand the overall mechanism of arsenicinduced carcinogenesis. A study presented during the meeting by Xie et al. investigated chronic oral arsenic exposure using mdr1a/1b(–/–) mice. They examined oxidative stress, altered gene expression, arsenic induced elevated lipid peroxidation products, and enhanced glutathione S-transferase (GST) activity in the liver. The study demonstrated that chronic arsenic toxicity, including oxidative stress, is enhanced in mdr1a/1b(–/–) mice, possibly due to accumulation of arsenic as a result of transport deficiency. Availability and solubility of a compound [ex: lead chromate (PbCrO4)] can determine its reactivity in cellular systems. Leonard et al. discussed their study on the effects of cellular exposure to PbCrO4 . These particles were used to investigate ROS generation and cellular response in RAW 264.7 cells. Cells exposed to PbCrO4 particles generated .OH from its endogenous H2O2 precursor, as measured by ESR spin trapping in combination with H2O2 and the ·OH scavengers, catalase and sodium formate. Confocal microscopy was used to show that these cells engulfed the particles over a 120 min time period, causing a significant increase in both H2O2 and oxygen consumption by the cells as well as activation of NF-κB and AP-1 transcription factors. The investigation demonstrated that once the PbCrO4 particles were engulfed by the RAW 264.7 cells they could induce chronic production of ROS through the reduction of Cr(VI) leading to the activation of NF-κB and AP-1. Residual oil fly ash (ROFA), which has also been found to generate ROS, was used in an investigation to determine of lung toxicity and its relationship to metal composition this study was reported at the meeting by Antonini et al. ROFA is a particulate pollutant generated by the combustion of fossil fuels and is made up of soluble and insoluble materials. The samples were separated into soluble and insoluble fractions and the metal composition was determined. ESR was used to detect short-lived free radical intermediates. Male SpragueDawley rats were instilled with ROFA. The results showed that ROFA, particularly the soluble fraction in particular generated a metal-dependent .OH radical. The soluble fraction was also shown to cause greater inflammation and result in reduced lung defense against infection compared with air heated ROFA. These results are most likely due to differences in metal composition and solubility of the ROFA samples. Copper poses a concern as an environmental hazard due to its persistent nature and ability to cause toxicity at high levels. Copper is capable of generating ROS to produce in-
tracellular oxidative damage. Mattie and Freedman reported their investigation on the ability of copper exposure to activate both metal and ROS-responsive signal transduction pathways. Their results demonstrated that copper did activate transcription via both metal and ROS-responsive pathways. They also showed that pretreatment with antioxidants such as vitamin E and aspirin provided partial protection. The results suggest that metal and oxidative stress-responsive signal transduction pathways mediate molecular and cellular responses associated with exposure to high levels of copper.
Metal-induced regulation of transcription factors Transcription factors are able to recognize and bind to specific sequences of DNA. This specificity is important in regulating the expression of target genes. Regulation and activation of transcription factors is an important element in mediating cellular responses to stimuli. Previous investigations show that exposures to metals such as Cr, Ni, V and Pb are able to activate cellular transcription factors in response to exposure. These transcription factors include NF-κB, AP1 and p53 [15, 16, 41, 45, 48–52]. ROS have been shown to play a role in certain transcriptional activations by metals but some pathways remain to be investigated. During the conference, Huang et al. presented their investigations into transcription activation by Nickel (Ni) compounds. The signal transduction pathways leading to transcription were studied in order to help understand the carcinogenic effect of Ni compounds. Ni compounds are a somewhat unique class of metal compounds in that they can induce transformation in cell culture as well as tumor formation in animal models. Their mechanism of action is not yet well understood. Exposure of cells to the Ni compounds NiS3S2 and NiCl2 resulted in the activation of nuclear factor of activated T cells (NFAT) but they did not show activation of p53 or AP-1. Ni compounds were also found to generate ROS and the addition of antioxidants such as N-acetyl-L-cysteine (NAC) and catalase, or the chelation of Ni with deferoxamine, resulted in the inhibition of NFAT activation. Sodium formate (an ·OH scavenger) or superoxide dismutase (an O2·– scavenger) did not inhibit NFAT activation. These results suggest that NFAT activation by Ni compounds appears to be mediated by H2O2. Ni exposure was also found to result in the transactivation of hypoxia inducing factor-1 (HIF-1), activation of phosphostidylinositol 3-kinase (PI3K), p70 S6 kinase (p70S6K), all of which have been shown to be important in tumor promotion and progression. Further studies show that Ni compounds induce HIF-1 transactivation through a PI3K/Akt-dependent and p70S6K – independent pathway. In addition to showing that PbCrO4 generated ROS Leonard et al. also discussed the ability of PbCrO4 to induce the tran-
6 scription factors NF-κB and AP-1. Cells phagocytized the particles leading to activation of NF-κB and AP-1. Such induction of transcription pathways may be involved in the inflammatory and carcinogenic responses induced by Cr(VI)containing particles. Cr(VI) can suppress both DNA replication and transcriptional as a result of Cr-induced DNA damage. In another study concerning Cr, Xu et al. reported their study on the identification of the molecular mechanisms involved in the reduction of RNA synthesis by Cr. Their results suggest that the suppression of RNA synthesis by Cr is related to chromium-induced DNA damage with prevents elongation and premature RNA polymerase arrest. Salazar et al. reported their study on the expression of the p53 protein was induced in the lymphocytes of healthy individuals and those in a chronic arsenic endemic region. The results generated suggest that there is a relationship between non-melanoma skin cancer and p53 expression in lymphocytes. The investigation demonstrates the use of expression of p53 in lymphocytes as a potential biomarker for susceptibility. Most studies involving Lead (Pb) focus on organ system effects even though its been shown to exhibit a high degree of toxicity. The molecular mechanisms by which Pb induces toxicity and carcinogenesis remain to be determined. As reported at the meeting, Tchounwou et al. used the CAT-Tox assay to assess the transcriptional responses associated with Pb exposure to 13 different recombinant cells lines generated from human liver carcinoma cells. The results indicated that Pb could cause cell proliferation, protein damage, growth arrest, and DNA damage. The results also showed increased induction of the GADD45 promoter and activation of the NFκB transcription factor, indicating a potential for involvement in oxidative stress.
Effect of metals on signal transduction pathways As discussed in the previous section, metals have been shown to activate transcription factors such as NF-κB, AP-1 and NFAT. However, it has also been suggested that metals affect the upstream regulatory components of the MAP kinase pathway. Different metals have been shown to affect the three major MAP kinases (ERK, JNK, and p38) through different pathways [15, 53–55]. Cr activates all three of the aforementioned MAP kinases in a dose and time dependent manner. Other metals may activate only one or two of the MAP kinases, or even cause one pathway to inhibit another [56, 57]. Crosstalk between the MAP kinase pathways is another important factor in determining the effects of metal exposure [58–60]. Activation of different pathways under altered conditions may contribute to determining whether a cell undergoes
apoptosis or necrosis [46, 61]. The role of ROS in these pathways is also being explored. The potential understanding of the importance of MAP kinase pathways is crucial to our study of metal-induced carcinogenesis. Vanadium is a metal, which is found throughout the environment. The toxic effects of vanadium and vanadate-induced phosphorylation of Akt and p70S6K were reported by Li et al. at the conference. These two kinases are involved in cell growth, transformation, and the transition of the cell cycle. Mouse epidermal JB6 cells were exposed to vanadium in a time and dose dependent manner. Vanadium caused translocation of atypical isoforms of PKC to the cytoplasm, suggesting that atypical PKCs (aPKC) are involved in vanadium-induced cellular response. The results suggest that signal transduction pathways leading to Akt and p70S6K activation/phosphorylation were different due to the differential role of aPKC in vanadate-induced phosphorylation of Akt and p70S6K. Another study concerning vanadate was presented by Luo et al. at the meeting. This group investigated the role of vanadate in apoptosis in cultured cerebellar granule progenitors (CGPs), since vanadate has been found to cause developmental defects in the central nervous system. Treatment of CGPs with vanadate showed activation of the ERK and JNK MAPK, but not the p38 MAPK. Vanadate also induced FasL production, the association of Fas with Fas-associated death domain (FADD), and the activation of caspase-8. While exposure to vanadate also generated ROS, they were found not to be involved in vanadate-mediated MAPK activation in the CGPs. Vanadate-induced FasL expression was ROS-dependent but JNK-independent, suggesting that JNK signaling plays a major role in vanadate-mediated activation of the FasFADD-caspase-8 pathway, which explains vanadate-induced apoptosis in CGPs. Arsenite stimulated IKKβ deficient cells were used to investigate the interaction between oxidative stress and JNK activation as reported by Chen et al. at the conference. Oxidative stress in cells during inflammation can induce activation of both IKK and JNK. Cross-talk between these two kinases is not well understood. Using wild type (WT) and IKKβ knockout (IKKβ–/–) mouse fibroblasts it has been found that IKKβ deficiency caused prolongation of stress-induced JNK activation in response to arsenite treatment. This extended JNK activation was found to be largely due to oxidative stress. An increased expression of p450 1B1 mRNA was observed in IKKβ–/– cells but not in WT cells. The results of this investigation demonstrate that increased p450 1B1 mRNA expression is an important factor in arsenite-induced JNK activation due to oxidative stress. Arsenic was also used to investigate into the role of MAP kinases in cell transformation and apoptosis as reported by Dong at the conference. Arsenite was found to induce phosphorylation of ERKs and JNKs in JB6 cells and induces AP1 and NF-κB activation. This metal, however, did not induce
7 p53-dependent transactivation. Blockage of NF-κB activation inhibited arsenic-induced apoptosis and enhanced arsenic-induced cell transformation. These results suggest that the activation of ERKs is required for arsenic-induced transformation and that the activation of JNKs and NF-κB are involved in arsenic-induced apoptosis in JB6 cells.
Metal exposure effects on apoptosis and cell growth regulation Apoptosis, or programmed cell death is a controlled response by which cells can die and yet have a minimal impact on the cells around them. It is genetically controlled involving embryogenesis, functional/immune regulation, and tissue homeostasis [62–65]. Apoptosis can be activated by a wide variety of signals and environmental factors, including bacterial toxins, viral infection, cell starvation, oncogene expression, exposure to oxidants, and UV radiation [47, 66]. Apoptosis serves the useful purpose of safely eliminating genetically damaged cells or cells with developmental errors from the tissues involved. Of interest to this conference, metals including As, Cd, Cr, Ni, and V have been found to induce apoptosis. Their mechanism of action remains to be elucidated. We have seen in previous studies that metals can cause apoptosis. During the conference, Beyersmann et al. reported their study on apoptosis induced by cadmium and zinc. Both cadmium and zinc produce morphological and biochemical effects which are features of apoptosis. The relationship of p53 with metallothionein (MT) induction and apoptosis was presented by Cherian and Fan at the conference. Human breast cancer epithelial cells, p53+ and mutated p53–, were exposed to cadmium or copper. The p53+ cells showed increased apoptosis and were sensitive to cytotoxicity of these metals. However, p53– cells were resistant to apoptosis and cytotoxicity of these metals. Their investigation showed a role for p53 on metal induced MT synthesis and apoptosis in human breast cancer epithelial cells. Protein kinase B (PKB)/Akt and its upstream signal transducer, phosphatidylinosito-3 kinase (PI3K) play an essential role in control of transcription and translation. Zhang et al. reported their study on the vanadate-induced activation of PI3K and Akt. Transcription factor E2F1 is a component of the downstream signals, which are regulated by Akt. The release of E2F1 results in a transition from G1 to S phase. It has been found that vanadate treatment increased the percentage of cells at S phase and triggered phosphorylation of pRb as well as the release of E2F1. After treatment with vanadate, it was found that inhibition of Akt decreased the percentage of cells in S phase and reduced both cyclin E and E2F1 expression and phosphorylation of pRb. This study demonstrated that Akt plays an essential role in the vanadate-induced
increase in cell number at S phase and transition from G1 to S phase through the E2F-pRb pathway. Ceryak et al. reported their study on Cr(VI)-induced apoptosis and/or cell cycle arrest via p53. The study sought to determine the effects of Cr(VI) transcriptional regulation and the role of ERK activation in cellular response. The findings suggested that both p53-dependent and p53-independent apoptotic and cell cycle pathways were affected by exposure to Cr(VI). The ability of Cr(VI) to affect important apoptotic and growth arresting genes appears to be independent of ERK.
Metal-induced mutagenesis and carcinogenesis The mutagenic and carcinogenic effects of arsenic were recurring topics at the conference. The molecular mechanisms of arsenic carcinogenesis were the subject of a presentation given by Huang et al. Numerous mechanisms have been proposed but the exact mechanism of action remains to be defined. Metabolism of arsenic may play a key role in the carcinogenic process. Arsenic toxicity research may be complicated by species specific differences in arsenic metabolism and toxicity. Individual sensitivity could be a result of differences in genes controlling metabolism, DNA repair, cell transport, immune response, antioxidant defense, and cell cycle control. ROS signaling pathways may also be involved in arsenic carcinogenesis. Other potential carcinogenic effects of arsenic include oxidative stress, genotoxic damage, DNA repair inhibition, and activation of signal transduction pathways. Shi et al. did another presentation, which investigated the oxidative mechanism involved in As toxicity and carcinogenesis. There is increased evidence of a correlation between generation of ROS, DNA damage, tumor promotion and arsenic exposure. The results suggested that oxidative stress plays an important role in the molecular mechanism of arsenic-induced toxicity and carcinogenesis. Exposure to arsenic was found to cause the generation of NO and O2·– which can subsequently be converted to more biologically damaging radicals such as ONOO– and .OH. These radical species can cause oxidative stress, lipid peroxidation, DNA damage, and activation of signaling pathways leading to tumor promotion and progression. Kitchen et al. also found that arsenic may proceed via ·OH radical production by a Fenton-like reaction or other means of free radical generation. Arsenic toxicity was examined from a risk assessment approach as reported by Tchounwou et al. at the conference. Recent reports have pointed out that arsenic poisoning has become a major health problem. Acute and chronic exposure to arsenic has been reported in several countries around the world and exposure through drinking water can cause bladder, kidney, skin, liver and colon cancer. It is therefore important to de-
8 velop a health risk assessment plan based on comprehensive analysis of the currently available scientific information as well as health guidelines and treatment technologies. The molecular biology of Cr(VI)-induced carcinogenesis was the subject of a presentation by Wise et al at the meeting. Cr(VI) is a well established cause of human bronchial carcinomas and fibrosarcomas. The genotoxic mechanisms of both insoluble and water soluble salt forms of Cr(VI) may be mediated by soluble Cr(VI) ions [48, 67]. This group developed a new model system of human bronchial cells by introducing hTERT, the catalytic subunit of human telomerase, into primary human bronchial fibroblasts. Because the telomerase doesn’t compromise growth or the response to Cr(VI), the results indicate that this could be an excellent system for studying the mechanisms of Cr(VI) and other potential carcinogens implicated in lung cancer development. Leonard et al. also reported on the activation of signal transduction pathways by Cr(VI) exposure which lead to inflammation and carcinogenesis. A possible model for Cr toxicity was presented by Ramsey and Dalal at the conference. Two complexes of Cr (4+) were presented as structurally well-characterized compounds of chromium in its redox active states. The synthesis procedure is outlined, and it is hoped that these compounds will provide a valuable resource to help in the definition and understanding of the biochemical mechanisms involved in chromate-related genotoxicity.
Future studies During the conference state-of-the-art information was presented and discussed by groups studying metal-induced carcinogenesis and its mechanisms. Metals ranging from arsenic to vanadate and their effects on cytotoxicity, cellular responses, apoptosis, cell cycle regulation, nuclear transcription factors, signal transcription pathways, mutagenesis, and gene expression were covered. Although the information presented demonstrated an increased understanding of the molecular mechanisms and modes of metal-induced carcinogenesis many questions remain to be investigated. As more research is shared and published many contradictory findings in the mechanisms involved in metal-induced carcinogenesis are being reported. An understanding of each metal, and differences in the investigated systems is needed to help resolve these issues. The role of ROS, the different metal metabolites produced and the pathways involved need to be clarified. The effect of metals on the cell growth cycle has also surfaced as an important area of research. Metals such as chromium are being better understood and models are under development to aid in further study. Signal transduction and transcription factors remain an important area for research in metal-induced carcinogenesis, as the differences in pathway activation and inhibition require further attention.
New techniques are becoming available to unravel the mechanisms of toxicity and carcinogenesis in more precise molecular terms. Intricate biological relationships are being investigated such as, biological components that influence the outcome of metal exposure, as well as activations of biological systems at the molecular, cellular, tissue, organ levels and whole animal levels. Techniques are being developed to examine cellular receptors, enzymes that can modify or activate metal toxicity, enzymes that repair DNA damage and factors that regulate cell cycle control. Genetic differences between individuals can affect the relative sensitivity of each individual to metal exposure. Complex interplay between multiple genetic and environmental factors on the affected genes can be important in determining this sensitivity. If we can identify and characterize these metal response genes, we will increase our understanding of human metal-induced disease susceptibility. Using the newly developed methods in microarray technology, we can better understand these relationships. Since microarrays depend upon hybridization single nucleotide polymorphisms (SNPs) can be detected. This knowledge could be used to protect individuals from metal-induced disease. These metal responsive genes are likely to fall into the following areas: cell cycle, DNA repair, cell division, cell signaling, cell structure, gene expression, apoptosis, and metabolism. More specifically, these new techniques include DNA microarrays, which with the elucidation of the human genetic code can function as a valuable investigative tool. DNA microarrays can assay large numbers of proteins and determine their response to metals. This method could also possibly be used to determine gene expression changes in genomic DNA after metal exposure. In addition to DNA microarrays the expansion of other avenues of technology may also contribute to our understanding of metal-induced carcinogenesis. With the sequence of the mouse genome almost complete, new knock out mice are a possibility, and can be used to elucidate interrelationships between metal activation pathways in vivo. RNA interference (RNAi) is another new method, which uses the synthesis of a complementary sequence to bind with cellular RNA. This process produces an effect similar to a knockout gene in that the RNA is generated by the cell but does not produce a protein because it is degraded prior to translation due to specific RNAse degradation. This method could be valuable in blocking specific metal responsive RNAs and measuring their effect. Toxicogenomics is a new field, which investigates how the entire genome is involved in biological responses of organisms exposed to environmental toxicants, such as metals. New methods based on advances in genomics have recently emerged. As a result, important questions that have long been unanswered are now open to investigation. These new techniques and methods can help define cellular networks of re-
9 sponse genes, identify targets of toxic response, provide biomarkers and testing protocols, and identify individuals with increased susceptibility to metal-induced carcinogenesis and disease.
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