Journal of Biotechnology, 16 (1990) 17-36

17

Elsevier BIOTEC 00540

Minireview

Environmental carcinogenesis and biotechnology Margaret M.L. Chu and Arthur Chiu Office of Research and Development, US EPA, Washington, DC, U.S.A.

(Received 26 October 1989; accepted 22 February 1990)

Summary Numerous environmental and host factors, some of which are known and some unknown, contribute to cancer development. While data and studies abound, our current understanding of the relation between cancer and the environment is still very limited. Understanding environmental carcinogenesis is critical to its effective management. Biotechnology has revolutionalized the study of biological and biomedical sciences. This minireview provides an overview of environmental carcinogenesis with emphasis on the contributions and prospects of biotechnology in advancing an understanding of environmental carcinogenesis for its prevention and intervention. Cancer; Environment; Biotechnology; Prevention; Treatment

Introduction

The intention of this minireview is to provide a brief overview of environmental carcinogenesis and the contributions and prospects of biotechnology in providing a better understanding of how cancer develops to support its effective management. Carcinogenesis is a complex and dynamic interaction of host and environment. Important host factors include genetic constitutions and health status. A m o n g environmental factors, diet, environmental pollutants, occupation and hfe-style

Correspondence to: M.M.L. Chu, RD-689, Office of Research and Development, U.S. Environmental Protection Agency, 401, M Street SW, Washington, DC 20460, U.S.A. * This article was written in the authors' private capacities and does not reflect the views of EPA.

0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

18 factors such as smoking have been implicated. Furthermore, cancer is believed to be preventable (Doll and Peto, 1981). Understanding environmental carcinogenesis is critical for its effective management. What must we know? What must be done? What is biotechnology's role? Cancer is an old malady. Paleopathologists have noted neoplastic lesions in dinosaur bones and autopsies of mummies have shown the existence of bone tumors (Zimmerman, 1977). There has always been a quest for knowledge and understanding of cancers, however advances have been limited by the experimental techniques available. Developments in molecular biology prompted biochemical and mechanistic investigations. The finding that some agents which damage DNA (e.g. radiation and reactive alkylating chemicals) can cause cancer has led to a focus on mutations as etiologic events in carcinogenesis. The studies of Bishop's group (1987) on retroviral genetics and molecular genetics of cancer has provided evidence at the molecular level of the involvement of genetic elements in cancer development. As our ability to study biological systems at the molecular level increases the evidence for cancer as diseases resulting from an accumulation of genetic damage also increases (Vogelstein et al., 1988). Significant progress has been made in our knowledge of inherited cancers and inherited conditions as genetic predispositions to cancers (Knudson, 1986; Cavenee, 1989). This progress is aided by studies of rare congenital chromosomal disorders and cancers (Rowley, 1973; Pathak, 1986; Pathak and Goodacre, 1986; Fong and Brodeur, 1987; Sandberg et al., 1988) which have provided markers for molecular studies at the gene level. The findings from inherited cancers support the current enthusiasm and need for the identification of critical target genes as well as the study of their structure, function, expression and, regulation of expression. However, one of the least understood areas of the development of cancers is the interaction between genetic and environmental factors. Traditional epidemiology and chronic animal studies investigate host and environmental factors in isolation (Tomatis, 1988), but understanding host-environmental interaction requires studying both in consort. This may account for the lack of mechanistic understanding of environmental carcinogenesis. More integrated multidisciplinary approaches in the study of putative genetic and erwironmental factors are needed. Developments in biotechnology are providing new tools to make such studies feasible. The following sections will present a concise synopsis of carcinogenesis, some of the molecular biological techniques for its study and the progress and prospects of molecular biologic techniques in improving our understanding of environmental carcinogenesis.

Carcinogenesis As stated in the introduction, understanding environmental carcinogenesis is critical to its effective management. Furthermore, understanding the pathogenesis of cancer from a clinical and experimental perspective is important for the identification of critical environmental and host factors for intervention.

19 The most common developmental pattern of cancers is their progressive potential for unrestricted growth. Major inquiries and treatment strategies have concentrated on this characteristic. Studies to date indicate that cancers have many causes, and their pathological development is multistage in nature. Furthermore, the responses of cancers to therapy are unpredictable. Theories of carcinogenesis can be dated to the 4th century when Hippocrates proposed that cancer was a disease of excess black bile. Association of environmental factors with cancer is not new. Ramazzini in 1700 ascribed the high occurrence of breast cancer among nuns to their lifestyle. John Hill suggested in 1761 that tobacco snuff is cause of nasal polyps. Percivall Pott in 1775 described the association of occupational exposure of chimney sweeps to soot and scrotal cancer. Johannes Muller in 1838 demonstrated that cancer tissue was made up of cells. Many hypotheses of the origin and development of cancer were presented during the 19th century. In general they can be described as the irritation, embryonal or parasitic hypothesis (Pitot, 1986a). Concepts of carcinogenesis continue to emerge in this century. They are in general attempts to describe the clinical, or experimental phenomenon. Boveri (1929) suggested that malignant tumors might be the results of a certain abnormal condition of the chromosomes, which may arise from multipolar mitosis. Our current findings of chromosomal conditions and cancers support Boveri's suggestion. Concepts about carcinogenesis are numerous and at times confusing. No coherent picture of carcinogenesis is available. The available concepts range from generalized mathematical expressions to mechanistic hypotheses (Iversen, 1988). Terms such as two-stage carcinogenesis, multistage carcinogenesis, initiation, promotion, progression, monoclonal, polyclonal, cocarcinogenesis, syncarcinogenesis, anticarcinogenesis, enhancement, synergism, genotoxic, epigenetic, oncogenes, cancer suppressor genes have been coined to describe research observations in experimental studies. Two main themes, namely stages in cancer development and factors contributing to the process, can be used to describe these concepts.

Stages of carcinogenesis Early experimental models, supporting the stepwise development of tumors, were based on Berenblum and Shubik's (1947, 1949) studies of cancer formation in mouse skin. They stated that there are at least two stages - initiation and promotion - in chemical carcinogenesis. Progression was first used by Foulds (1957, 1958) to describe his observations of changes during advancement of neoplasms. Nowell (1976, 1986) further described tumor progression as developing in a stepwise fashion through qualitatively different stages. He also suggested that genetic instability was the fundamental mechanism responsible. These sequential changes include cellular morphology and behavior; increase in growth rate; escape of local growth control; continual proliferation in place of terminal differentiation or death; metabolic alterations; decreases in antigenicity and acquisition of drug resistance. In addition he also presented evidence in support of a clonal evolution

20 concept of cancer development (Nowell, 1986). Ling et al. (1985) studied the genetics of tumor progression and their findings support the concept of genetic instability as a cause in cancer development. The instabilities they observed were characterized, among other genetic alterations, as chromosomal breaks, non-disjunctions and ploidy changes. Farber's studies of hepatocarcinogenesis in the rat (Farber and Cameron, 1980; Farber and Sarma, 1987) have provided the most detailed information available concerning the stepwise development of tumors in animals. They characterized the development of neoplasms in stages and indicated that there are steps of biological events within the stages of initiation, promotion and, progression. Studies of cellular and molecular events using mammalian cells in culture also provide evidence for stepwise changes (Weinstein, 1985; Nettesheim et al., 1987; McCormick and Maher, 1989; Weinberg, 1989). These studies provide evidence for the multistage nature of carcinogenesis at the molecular level. It should be recognized that these stages/steps are linked to the experimental design for exploring the mechanisms of carcinogenesis. Even though some of these stages/steps are well characterized, their relevance to human cancers is presently uncertain. Early statistical analysis of age-cancer incidence curves also suggested stages in cancer development (Nordling, 1953; Armitage and Doll, 1954). Nordling (1953) observed that most adult cancer mortalities show a steadily increasing incidence which follows a high power of time. He further suggested that such a curve could be explained by cancer originating from a single cell that had sustained a number of mutations. Armitage and Doll (1954) analyzed the changes of site- and sex-specific cancer mortality data with age and concluded that the results accorded with Nordling's hypothesis. They suggested that carcinogenesis is complex and the data can be explained by a process with six or seven stages, but these stages are not necessarily mutations. Shortly after they proposed the multistage model for analyzing cancer incidence data, Armitage and Doll (1957) proposed a two-stage theory for the age distribution of cancer which incorporated the idea of clonal growth. Knudson (1971), 14 years later, analyzed information on 48 cases of retinoblastoma and proposed a two-mutation, rate-limiting event model. Further analysis of human cancers has led to the proposal that a two-stage model with clonal growth may have general applicability (Moolgavkar and Knudson, 1981; Knudson, 1987). More recently, Gaffney and Altshuler (1988) analyzed the Doll and Hill data on smoking and found that a two-stage model with clonal growth fitted the data better than a multistage model. Even though the number of stages vary as the cancer, host, and environmental factor varies, it seems reasonably clear that human and animal cancers develop in stages. It is now appreciated that cancer development in humans and animals normally spans a substantial fraction of their lifespan; this period of time or latency must occur before the disease is expressed. The developing pathology is accompanied by progressive molecular, cellular and tissue changes (Farber, 1982; Pitot, 1986b). The biological events or phenotypic cellular changes observed are described as the stages of carcinogenesis.

21 These stages are further described as initiation, promotion and progression. Uniform definitions for these terms are not available because they are linked with specific experimental procedures or pathological interpretations. In general, and for experimental systems in particular, the term initiation is used to describe early changes in cancer development associated with damages to DNA. Promotion as a stage is less well understood, but is usually used to describe events subsequent to D N A damage and is associated with cell proliferation activities. Progression is used to describe the last stage of cancer development, and is characterized by increase in chromosomal/karyotype changes and progressive ability of the cell to escape normal control of growth and differentiation. This stepwise development of cancer is generally referred to as the multistage concept of carcinogenesis with the major stages characterized as initiation, promotion and, progression (Pitot, 1986b; Farber and Sarma, 1987).

Factors contributing to carcinogenesis Much of our interest in cancer is stimulated by a search for its causes and cures. The identification of factors contributing to cancers relies heavily on epidemiology studies and animal toxicity testing. The belief that cancers are avoidable led to intense efforts in search for environmental factors that contribute to its development. This search led to the identification of radiation, viruses and chemicals as causative agents, and dietary habit and life-style as h0st-related factors (Pitot, 1986b; IARC, 1987a). The most intensive effort has been devoted to the identification of industrial chemicals that can induce cancer. The International Agency for Research on Cancer (IARC) evaluated some 628 agents, industrial processes, occupational exposures and cultural habits for human carcinogenic risk (IARC, 1987a). IARC identified about 50 of these as human carcinogens; in addition, inadequate data were available to evaluate some 381 agents for possible carcinogenicity. Further, it has been estimated that adequate data are available to evaluate less than 1% of the chemicals currently available in commerce. It is apparent that our current methods for carcinogen identification have very limited capacity to deal with the large number of available chemicals. Chemicals only represent one of the many environmental factors that can affect cancer development. Doll and Peto (1981) divided life-style and other environmental factors into 12 categories and analyzed each category's relation to the trends of cancer deaths in the United States. They concluded that about 30% of the U.S. cancer deaths can reliably be ascribed to tobacco smoking, and dietary factor(s) may eventually be found to be of comparable importance. Increasing efforts should be expanded to understand the contribution of other environmental factors associated with cancers. The recognition that not all exposed individuals develop cancer indicates that genetic determinants may play an important role in cancer development. The significance of the genetic contribution has been difficult to determine. Before the

22 advent of biotechnology, family pedigree studies were a means to determine if genetic factors are involved in cancer development. One of the most rigorous approaches is to study twins [e.g., Cederlof et al.'s (1970) study of smoking and lung cancer]. Knudson (1986) has stated that, with rare exceptions (e.g., retinoblastoma), genetics is not the sole determinant of cancers. He argued that, while the majority of human cancers probably result from interactions of genetic and environmental factors, some cancers may be induced by environmental factors alone. Examples of such environmental factors may include the human carcinogens (e.g., vinylchloride) identified by IARC (1987a). The identification of host factors that modify susceptibility to carcinogens, and the interplay of host and environment is timely considering the advancements made in biotechnology. Chemicals can cause such chromosomal aberrations as breaks, deletions, or translocations (IARC, 1987b). Using automated equipment, modern cytogenetic techniques can detect such chromosomal changes rapidly. Chemicals also cause mutations in various organisms and cells (IARC, 1987b). The specific nature of these mutations, especially if it is a single nucleotide change, has, however, been difficult to pin down. Cloning and sequencing are tools derived from biotechnology which make the identification of single nucleotide change feasible.

Methods from biotechnology The power of biotechnology, 'industrial biology' or the 'new molecular biology' is in the provision of tools through the manipulation of nucleic acids to supply genetic reagents. The ability to identify, and isolate genetic elements, propagate them at will outside the host, and subsequently reincorporate these back into a host, gives us the ability to study life processes not achievable before. Burck et al. (1988) have summarized the tools of biotechnology that are available for the study of cellular and molecular events in carcinogenesis. Techniques routinely available for cellular and molecular studies include the use of: restriction enzymes; restriction mapping, DNA cloning; probes; Southern blotting; DNA sequencing; filter bound RNA assays; in vitro translation assays; polyacrylamide gel electro° phoresis; immunoassays; immunoblots (Western blots); cultured cells and phenotypes; NIH 3T3 cells and transformation; transfection and assay using intact organisms. Based on this list, it is apparent that there is a rich diversity of methods available for the study of cellular and molecular events in carcinogenesis. However, new and/or revised methods are being rapidly developed and no text can be current or comprehensive in this respect. Gene mapping, DNA sequencing, cell fusion, and transgenic techniques are briefly described below.

Mapping Mapping the chromosomal location of a gene is the necessary starting point for: gene isolation or cloning, the identification of their products, the study of their

23 mutations induced by environmental agents, and the study of the roles of these mutations in specific cancers. The human genome contains between 30000 to 50000 genes, thus gene mapping is a painstaking task (Frezal, 1987). When chromosomal changes are known they can serve as markers for the mapping of involved genes. Chromosome banding and other cytogenetic techniques are used to determine the chromosomal region for gene mapping. Catalogues of chromosome alterations in cancers (Mitelman, 1983, 1985, 1986) are available. These may be used to direct the mapping. Chromosome walking or jumping can be used to locate a gene on a chromosome. Chromosome walking is starting at a reference point on a chromosome and proceeds step by step in stages, or paces not exceeding the dimensions of an insert. Chromosome jumping proceeds from one point on the chromosome to another by jumps whose length depends on the enzyme used to cleave the chromosome and the frequency with which it induces cleavage (Rommens et al., 1989). Whatever method is employed, the goal is to map the location of a gene on a specific chromosome for cloning. There are different kinds of gene maps. A restriction map shows the order and distance between cleavage sites of site-specific endonucleases. Linkage maps are based on linkage between two genes at polymorphic sites giving rise to RFLPs (Restriction Fragment Length Polymorphisms) or VNTRs (Variable Number of Tandem Repeats). Contig maps represent the structure of contiguous regions of the genome by specifying the overlap relationship among a set of clones. Physical maps give the physical location of a gene on a chromosome. Many methods for mapping are currently employed and new ones will emerge (Olson et al., 1989). A low resolution genetic linkage map of the human genome is currently available (Donis-Keller et al., 1987). With the human genome project now in progress, it is projected that an intermediate resolution physical map of the human genome will be available in about 5 years (Olson et al., 1989). The effort will also provide new techniques in addition to information critical for the study of cancers.

DNA sequencing Once the gene of interest is located, the next step is to isolate and clone the gene for detailed structural and functional analysis. Sequencing is used to determine the nucleotide sequence of a gene or a segment of DNA. The first direct D N A sequencing technique was developed by Sanger in 1975 and has been subsequently improved (Sanger et al., 1977); other methods have been developed (e.g., Maxam and Gilbert, 1980). Automated procedures, such as those of Wada (1987), have resulted in DNA sequencing becoming a - relatively - routine procedure. In addition, optimization methods (Kambara et al., 1988) have been developed and computer software and hardware are available to analyze and store the sequenced data. The coupling of biotechnology and microelectronics has resulted in much more efficient methods for dealing with previously tedious tasks (Bishop and Rawlings, 1987). Polymerase chain reactions (PCRs) are a powerful method for in vitro gene amplifications (Saiki et al., 1988). PCRs can amplify the D N A from one cell by

24 factors ranging from a million (20 cycles) to a billion (30 cycles) and sequencing can be done without cloning the gene as a separate step which can take weeks.

Cell fusion Cell fusion is the key to the development of monoclonal antibody technologies (Kohler and Milstein, 1975). One technique involves fusing a myeloma cell line with a mortal B cell to give a 'hybridoma' cell which is immortal with respect to antibody production. Monoclonal antibodies are reagents used in enhancing the ability to purify and isolate gene products. Because of their high specificity and sensitivity, monoclonal antibodies are used in various sensing and monitoring procedures (e.g. the biomonitoring of pollutants). Furthermore, monoclonal antibodies have various clinical applications, including their use in the delivery and monitoring of drugs as well as the use of monoclonal antibodies as drugs in their own right: immunotoxins as antitumor reagents (Vitetta et al., 1987). Cell fusions or somatic cell hybrids are important methods for the study of recessive genes associated with cancer development. The identification of tumorsuppressor genes relies heavily on the use of somatic cell hybridizations. Gene transfers and transgenic animals The true novelty of biotechnology may well be the ability to exploit the universality of the genetic code to combine, in one organism, traits developed by organisms that have evolved along separate phylogenies. The production of transgenic animals via gene transfers is an example of such 'novelties'. Transgenic technology offers several advantages. In contrast with typical cellular and molecular studies, the transgenic animal can have functioning neural, immune and endocrine systems, as well as other biological control systems. Thus, possibly, insights into the impact of carcinogens on maintenance and regulation of homeostasis, where genetic elements are not the direct targets, can be studied in these animals. Jaenisch (1988) stated that the information gained from the use of transgenic technology is relevant to all aspects of modern biology including developmental gene regulation, the action of oncogenes, the immune system, and mammalian development. Genes can be introduced either into embryonic or somatic cells. Methods for gene transfer into cells and the strengths and limitations of the methods have been reviewed (Cline, 1987; Jaenisch, 1988; Orkin and Williams, 1988; Friedman, 1989). The techniques can be described as physical or biologic-vector-mediated. Physical methods include chromosome mediate transfers, transfection via calcium phosphate precipitates or electroporation, fusion of DNA in lipid or spheroplast membranes, and microinjection of DNA solution into nuclei of target cells. Both DNA viruses and RNA viruses have been used as biologic vectors for gene transfers. Microinjection (Gordon and Ruddle, 1983) and retroviral transfer (Kohn et al., 1987) are used most frequently due to the relatively high level of gene incorporation using these methods. Techniques used in the production of transgenic mice include: microinjection of eggs (Brinster et al., 1985); retrovirus infection of embryos or embryonic stem cell transfer into blastocysts which are methods of choice (Jaenisch, 1988).

25 Transgenic mice, with an oncogene such as ras, are commercially available. Such transgenic animals, which carry an activated oncogene in both germ and somatic cells, offer a shortened path for carcinogenesis studies. One day we may have models for carcinogen testing that are mechanism- and chemical class-specific. Gene transfer is the foundation for the development of gene therapy.

Progress Significant progress has been made in our knowledge of inherited cancers and inherited conditions and cancers (Fong and Brodeur, 1987; Hansen and Cavenee, 1987; Cavenee, 1989; Knudson, 1989). Progress in experimental systems is also impressive. We know much more about cell growth, cell differentiation, gene activation and inactivation, gene structure and function, cellular communications, elaborate systems of signal transductions that may be the key to understanding carcinogenesis (Bishop, 1987; Klein and Klein, 1987; Weissman et al., 1987; Bourne, 1988; Klein, 1988; Weinberg, 1989). These are the results of the contributions of the new molecular techniques. While the knowledge of inherited cancer supports the genetic origin of rare cancers, the molecular bases for these are largely unknown (Cavenee, 1989). For retinoblastoma, however, the mutation of a recessive gene is firmly established as the cause (Cavenee et al., 1985; Hansen and Cavenee, 1987). Knudson's (1971) analysis of the inherited form, as compared to sporadic cases of retinoblastoma, suggest a two-mutation event model. The studies of Cavenee et al. (1985) and Bookstein et al. (1988) definitely support this concept. Much progress has been made in understanding the role of the Rb gene in these cancers (Squire et al., 1986; Wiggs et al., 1988). Furthermore, the possibility of using DNA polymorphism for predicting the risk for hereditary retinoblastoma has been suggested (Bookstein et al., 1988). Studies of colorectal cancer, on the other hand, suggest that a multistage process occurs in at least some human cancers. The observed genetic changes of colorectal tumors (Fearon et al., 1987, Vogelstein et al., 1988) support a model in which accumulated alterations, affecting at least one dominantly acting oncogene and several tumor-suppressor genes, are responsible for the development of the tumors. The progressive nature of the genetic alterations, as the clinical disease progresses, further supports the participation of these cellular genes in colorectal cancer. These results can be used to direct the management of colorectal cancers through early detection and intervention. Efforts are currently being devoted to characterize further predisposing factors, such as familial adenomatous polyposis coli (Bodmer et al., 1987; Cannon-Albright et al., 1988). The stages of cellular and molecular changes can be used as disease markers for monitoring (Lipkin, 1988). It is believed that early detection, through screening, can reduce cancer mortality (Kinzie et al., 1988). Cellular and molecular biology of cancer Detailed investigations of many aspects of carcinogenesis that were not previously possible, are now feasible using techniques that involve one or more of the

26 following: cloning, sequencing, immunology, cell fusion and cell culture. Results obtained in the last decade suggest that cellular genes are molecular targets of multistage carcinogenesis (Bishop, 1987; Stanbridge, 1987; Weinberg, 1989). Proto-oncogenes or oncogenes may be defined as cellular genes which, when activated qualitatively or through quantitative alterations of their expression, contribute to the development of a malignant phenotype (Klein and Klein, 1986; Bishop, 1987; Weinberg, 1989). Tumor suppressor genes, anti-oncogenes, or recessive oncogenes, can be defined as negative genetic regulators of normal cell growth which, when inactivated through a lost or altered function, contributes to the malignant phenotype (Klein, 1987; Stanbridge, 1987; Barrett, 1988; Weinberg, 1989). Cell culture transformation systems, for example, have been used in screening for carcinogens (Heidelberger, 1980; Heidelberger et al., 1983). Transformation of cells can be described as the acquisition of one or more characteristics of tumor-derived cells. Malignantly transformed cells may be defined as those cells capable of inducing progressively growing, invasive tumors in a suitable host (McCormick and Maher, 1989). Transformation studies have made substantial contribution to our current concepts of the role of simple mutations, chromosomal alterations, DNA methylation, differentiation, oncogenes, tumor suppressor genes, growth factors, and tumor promotion in the process of multistage carcinogenesis. Sentinel phenotypes of presumed importance include alterations in morphology and growth pattern, and the acquisition of unlimited proliferative potential ('immortality') in culture, the ability to grow without attachment to a solid matrix (anchorage independence), and altered requirements for growth/nutritional factors (Boreiko, 1988). Treatment

The treatment of cancers has met with variable degrees of success. In general, when the pathogenesis of a cancer is well understood, the rate of success in its management is high. However, the development of drug resistance has made the design of therapy difficult. The knowledge gained on the molecular and cellular bases of carcinogenesis, coupled with new technologies, may provide new avenues of therapeutic approaches. These include the development of immunotoxins against tumors (Vitetta, 1987), interleukins (Smith, 1988; Hawkins, 1989), growth and differentiation control agents (Pierce and Speers, 1988), oligonucleotides as inhibitors (Stein and Cohen, 1988), tumor necrosis factors, and combination therapies (Krosnick et al., 1989). Leukemia is an example of how a firmer understanding of the multistage progression of cancer has contributed to cancer treatment. Clinically, it is now firmly established that myelodysplasia is a precursor of leukemia. This preleukemia can be recognized by the anemic state, other secondary signs and symptoms of cellular elements in the blood suggesting aberrant behavior of the myelopoietic stem cell. These signs are used as indicators for staged treatment protocols (Estey et al., 1987; Verwilghen and Boogaerts, 1987).

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Prospects With our tools advancing rapidly and the molecular and cellular determinants of cancer unfolding at an impressive speed, a better understanding of environmental carcinogenesis is envisioned. This may require modification of current approaches in the study of environmental carcinogenesis. Understanding interactions of genetic and environmental factors holds promise, and current advancements make it increasingly possible to perform studies using human systems. Moreover, it has been suggested that we already have a wealth of genetic information for human molecular genetic studies (White and Caskey, 1989). They stated that the available catalog of human genetic variants now describes more than 4000 distinct Mendelian conditions; by comparison, only 700 genetic loci have been identified in the mouse. Harris (1987) has also commented on the unique opportunity available to use human tissues and cells for carcinogenesis studies. Central to the study of cancers in humans, is the problem of extrapolating animal data to humans. It is recognized that molecular and cellular studies using human tissues also have their own set of limitations. The key limitation, typically, is detachment from a complete hierarchy of biological control. The emergence of transgenic animals as models should be able to bridge this gap. With this in mind, and the wealth of characterized human mutant phenotypes and molecular and cellular techniques at hand, it seems fitting to be optimistic that the riddle of environmental carcinogenesis may soon be further clarified. Areas that can be more effectively studied include ecogenetics and molecular epidemiology.

Ecogenetics Ecogenetics is the study of human genetic variation in response to environmental agents while pharmacogenetics is reserved for medical agents. Early ecogenetic studies were limited, as molecular methods were not available. Mulvihill (1976, 1984, 1985; Mulvihill and Tulinius, 1987) has discussed the attempts that have been made to compare various ecological and familial factors including the study of lung cancer and smoking as an example, of ecogenetic studies. Various techniques based on genetic polymorphisms (Gusella, 1986), should overcome some of the technological difficulties. Polymorphism among different indviduals and populations offers unique opportunities to understand multiple, simultaneously occurring interactions between genes and the environment, and the subsequent phenotypic expression of heritable characters. Early investigations (Kellermann et al., 1973a, b) of the inducibility of AHH (arylhydrocarbon hydroxylase) and lung cancers, indicated that a single locus with 2 alleles controls AHH induction. Kellermann et al. (1973a, b) found about 96% of the patients with lung cancers had high to medium inducibilities compared to 55% in controls. Our current knowledge of cytochrome P-450s and their role in xenobiotic metabolism and disease susceptibilities is quite elaborate (Nebert et al., 1982; Adesnik and Atchison, 1986; Gonzalez et al., 1986; Whitlock, 1986; Guengerich, 1988). This information supports the role of identifying polymorphisms to de-

28 termine differential susceptibilities for environmental carcinogens such as polycyclic aromatic hydrocarbons or drugs such as debrisoquine (Knudson, 1989). The pharmacogenetics of N-acetylation was studied by Weber and Hein (1984). They found some slow acetylators are homozygous for a 'slow' allele. Karakaya et al. (1986) studied patients with bladder cancer in order to determine their acetylator phenotypes for N-acetyltransferases. They found a correlation between the slow acetylator phenotype and bla~Jder cancer risk. The polymorphic expression of acetylation activities may be important risk factors in human susceptibility to bladder cancer from arylamine carcinogens (Kirlin et al., 1989; Knudson, 1989). The phosphorylation of proteins is a major mechanism of biological regulation and its biochemistry has been extensively studied (Krebs, 1985). Molecular studies have shown the existence of a large family of kinases (Hunter, 1987). Some of these kinases are involved in signal transduction pathways (Bourne, 1988). Furthermore, a linkage to oncogene activation has been implicated (Fry et al., 1986; McCormick, 1989). Understanding the ecogenetics of protein kinases and G-proteins may provide critical information for environmental carcinogenesis. Biotechnology allows the characterization of hereditary traits affecting carcinogen metabolism at the level of DNA. Gene identification, isolation and cloning can be used to determine whether gene modification, deletion, duplication and other such events have taken place that affect toxification and detoxification. The study of polymorphism may shed light on species differences in the metabolism of carcinogens and, in turn, species differences in response to these agents. Incorporation of appropriate ecogenetic diagnostic services into the general genetic services may be useful for public health monitoring in the prevention of environmental cancer.

Molecular epidemiology Tomatis (1988) stated that an impediment in the study of environmental cancer risks is that epidemiologists and clinicians have gone their separate ways without attempting to integrate their knowledge and approaches. Taylor (1989) stated that the problem is, that the study of human genetic susceptibility to environmental toxins has been split between two fields: genetics and epidemiology. Geneticists have investigated 'susceptibility' through family based studies of phenotype, while epidemiologists have investigated environmental factors using population-based questionnaires. Few studies look at both factors simultaneously. Molecular epidemiology is a term used to describe studies where molecular laboratory methods are used in analytical epidemiology (Harris et al., 1987). Perera and Weinstein (1982) discussed the prospect of molecular cancer epidemiology for carcinogen identification. Ideally, all interactions between the environment and the host should be investigated. These include the study of: host factors that influence susceptibility to carcinogens; detecting carcinogens in human tissues, fluids, and cells; measuring early biological events that are casually linked to exposure. Perera and Weinstein's efforts concentrated on the detection of carcinogens in human fluid using carcinogen-DNA adducts as a dosimeter (Perera, 1987). While the identification of carcinogens in human fluids has received much attention, the study of effects

29 at the molecular levels has been slow. This is because not much is known at the molecular level about carcinogenesis. New molecular techniques can be applied in epidemiologic studies resulting, hopefully, in studies that can be done with fewer subjects in a shorter time, and look at genetic and environmental factors simultaneously. Molecular techniques may also serve as a basis to bring a multidisciplinary team of scientists to advance our understanding of environmental carcinogenesis. The use of highly sensitive molecular techniques, such as the polymerase chain reactions, allow the detection of specific mutations with a limited amount of biological material. Such molecular biologic methods have the potential to identifying exposures to specific putative environmental agents that traditional epidemiologic approaches could not delineate. Somatic cell gene therapy The most attractive prospect for the application of biotechnology may rest in the development of human somatic cell gene therapy (Ledley, 1987a, b). This is based on the assumption that cancer results from genetic defects and definitive treatment for genetic diseases should direct the treatment at the site of the defect - the mutant gene - rather than the mutant gene product. Gene transfer techniques are fundamental to the development of human gene therapy. Researches from the National Institutes of Health, for example, have used lymphocyte-activated killing (LAK) cells and tumor-infiltrating lymphocytes (TIL) to treat high-grade malignancies. A technique was developed to isolate TIL cells from the patient, grow them in vitro with neomycin-resistant genes inserted; when the TIL cells are reincorporated for treatment the inserted gene serves as a marker to monitor therapy progress. While not a gene therapy itself, this technique can be viewed as the precursor of such. Aspects of the potential, progress, and prospect of human gene therapy have been reviewed (Caskey, 1987; Cline, 1987; Orkin and Williams, 1988; Friedman, 1989). Strategies for gene therapy include gene replacement, correction, or augmentation. Gene replacement therapy involves removal of a mutant gene and replacement with a normal gene. Gene correction requires restoration of the normal gene structure without changing other nucleotide sequences. Gene augmentation is the modification of the content of expression of mutant genes in defective cells. A number of methods have been developed to introduce functional genes into mammalian cells (Dick, 1987; Kohn et al., 1987; Orkin and Williams, 1988; Friedman, 1989). Prevention Cancer prevention may be achieved through modifications of genetic factors that predispose to its development and by modulating environmental factors which induce the process. Although human gene alteration as a strategy for modulating genetic predispositions is not available now, biotechnology has the potential of making these techniques a reality in the future. It is our opinion that modifying

30 genetic factors in humans should not be a primary strategy in the prevention of cancers.

We are at a time akin to a new industrial revolution. A revolution characterized by high-speed computers and industrial biology. An optimistic perspective is that biotechnology m a y alter the non-genetic host factors and the environmental factors associated with cancer development and provide new therapies (Chu and Kameley, 1988). Modifiable host factors include dietary habit and life-style. Environmental factors may be modified through changes in industrial, agricultural and waste management practices (Lindow et al., 1989). Bioprocessing can be used in the production of goods and commodities, biomass as source for energy and chemicals (Lipinsky, 1981; Stiefel, 1987), biotechnology-based agriculture and biological treatment of wastes (Lal et al., 1984; Glick and Skof, 1986; Buswell and Odier, 1987). As stated earlier, a third of the cancers in the United States are related to tobacco smoking, and these, of course, can best be prevented by not smoking. Another third of U.S. cancers are related to diet, and biotechnology m a y possibly play a role in modifying the nutrient contents of our food supply to ensure a palatable diet that is low in cancer risk factors. Similarly, improved and disease-resistant crops and the use of biopesticides may minimize exposure to hazardous chemicals. And disease-resistant live-stock may lower drug residues in meat. The introduction of nitrogen-fixing plants may reduce the need for chemical fertilizers. The application of microbes in waste treatment is not new, but new techniques may widen their application. The use of microbes for the removal of waste such as persistent organic toxins, pesticides and lignin have been reviewed (Kobayashi and Rittmann, 1982; Lal et al., 1984; Hwang et al., 1987; Rehacek and Krumphanzl, 1987). An interesting application is the use of microbes in mining (Toma, 1988). New technology, however, brings unknown risks, such as the introduction of deleterious traits into the environment. While the potentials of the new technologies are great, experiments and applications must be carefully planned to take into account the potential for harmful consequences to the environment (Chu and Kameley, 1988; Lindow et al., 1989). An organism released into the environment m a y adapt, giving ecological advantage, to harm the environment.

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