Environmental Biomonitoring - ACS Publications - American Chemical

Chapter 16

Biomarkers of Inherited and Acquired Susceptibility to Toxic Substances

Downloaded by STANFORD UNIV GREEN LIBR on October 1, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0654.ch016

E. Nieboer Department of Biochemistry and Occupational Health Program, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada

A brief overview is presented of genetic testing within the context of molecular epidemiology. Results are outlined for three pertinent studies completed in the author's laboratory: (1) the relationship of gastrointestinal absorption of aluminum to Alzheimer's disease and the allelic risk factor apolipoprotein E-ε4 for the latter; (2) inherent metallothionein induction capacities in peripheral-blood mononuclear leucocytes of non-smokers; and (3) characterization of nickel-induced mutations to illustrate compound-specific, acquired genetic damage. Potential applications in genetic screening (i.e., the detection of inherited susceptibility/protection factors) and genetic monitoring (i.e., determination of acquired changes in an individual's genetic material) are discussed.

Genetic testing may be subdivided into genetic screening and genetic monitoring (7,2). The emphasis in genetic screening is to detect inherited traits that predispose an individual to a pathologic effect if exposed to toxic agents (7-5). By contrast, genetic monitoring involves periodic examination of individuals to evaluate modifications within the genetic material that evolve over time, perhaps due to occupational or environmental exposures (7,2). Chromosomal damage or increases in DNA mutations might be the measured outcome (4). Within the context of the human genome project (5), with the goal of mapping and sequencing the entire genome, the application of genetic testing technologies in assessing the impact of exposures to both genotoxic and non-genotoxic agents may be expected to become routine. The objective of the current presentation is to share the result of three studies in the author's laboratory that illustrate this expectation.

© 1997 American Chemical Society

In Environmental Biomonitoring; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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EXPERIMENTAL CONSIDERATIONS


GI Absorption of Aluminum in Healthy Volunteers and Alzheimer's Patients A critical review of the available literature suggests that age, Alzheimer's Disease (AD) and renal disease appear to enhance the gastrointestinal absorption of aluminum (6). Increased absorption with age and in patients with AD who were 76 years or younger was reported by Taylor et al. (1992). They assessed the increase in serum aluminum 60 minutes after administering an oral drink of aluminum citrate. On the basis of these findings, we hypothesized that the presence of the apoE4 allele (apolipoprotein, genetic variant ε4) might correlate with an increased capacity to absorb aluminum from the GI tract. The apoE4 allele appears to constitute an important risk factor for AD (7,8). Serum samples for aluminum analysis (by electrothermal atomic absorption) were obtained from 20 healthy volunteers (age 15-59), 10 probable AD patients (age 64-84) and 7 healthy age-matched controls. For the AD patients, a blood sample was also taken and sent to the IMAGE Projet for apoE4 genotyping (see Acknowledgements). Immediately after these collections, 300 mg of aluminum (13.5 mL of aluminum hydroxide as Amphojel suspension) and 4.8 g of citrate in 100 mL of diluted lemonaid were ingested. One additional tube of blood for serum aluminum analysis was drawn 90 minutes after the ingestion of the aluminum/citrate solutions. Although a full statistical analysis of the data has not been completed at the time of writing, a number of tentative observations are rendered, (i) All aluminum levels before the Amphoj el/citrate ingestion are in the accepted reference interval of 1-6 μg/L. (ii) The age-dependence of GI absorption of aluminum is not readily apparent, (iii) About 20% of all donors could clearly be considered "high absorbers", with final serum aluminum levels exceeding 150 μg/L, and this is independent of age. (iv) Six of the ten probable AD patients has at least one copy of the apoE4 allele, with a prevalence of 35%. (v) Even though the number of AD patients is small, there is no apparent link between aluminum absorption and the presence of apoE4 (only 1/10 had a final serum aluminum level > 150 μg/L). Our preliminary findings favour the interpretation that the weak epidemiologic link observed between AD and aluminum intake from drinking water (6) may well reflect a positive bias in the clinical diagnosis of AD. The latter may be due to independent neurologic symptoms arising from enhanced aluminum uptake in a significant proportion of individuals of AD age. One might also speculate whether the "high absorber" phenotype has a genetic component. Metallothionein Induction in Peripheral-Blood Mononuclear Leucocytes of Nonsmokers and Smokers Besides roles of metallothionein (MT) in essential metal metabolism (10), it is


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generally accepted that elevated levels of MT can protect cells from acute exposure to metals (especially zinc and cadmium) or even organic species (including reactive electrophilic agents) (11). Since MT is an inducible protein, a pertinent question is whether inter-individual differences exist in MT expression. In 1989, we observed that MT induction capacity varied considerably in cells from 39 healthy, non-smoking volunteers aged 20-40 years (9). This was achieved by measuring both MT protein and MT messenger RNA (MT mRNA) in peripheral-blood mononuclear leucocytes (about 85% lymphocytes and 15% monocytes) before and after exposure for 12 or 6 hours to 10 μΜ CdCl in culture. While the induction capacity of MT mRNA, defined as the ratio of induced levels observed in the in vitro incubation to constitutive (basal) levels, varied about 10-fold, the corresponding factor for MT protein was 2.6.


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Recently, we have extended these studies to 20 smokers, aged 19-25. As before, basal levels were compared with in vitro concentrations induced during 16 hours with either 10 μΜ CdCl or 125 μΜ ZnS0 . The MT-protein induction capacity using CdCl varied 2.7-fold in the smokers, compared to 1.7-fold for the pooled variation in 2 non-smokers whose mononuclear cells were sampled 13 times over a 4-month period. Differences in variances between the two induction-capacity categories were statistically significant according to the two-tailed F-test (p< 0.025). For MT induction in mononuclear cells with zinc, the corresponding differences in induction capacities were not significant (a 2.0-fold range for the smokers and 1.5-fold for the intra-individual comparison). The interpersonal variation in basal, Cd-induced and Zn-induced MT concentrations ranged one order of magnitude. It is concluded that smoking does not appear to affect constitutive MT levels nor the MT-induction capacity in human peripheral-blood mononuclear cells. 2

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Characterization of Nickel-Induced Mutations in AS52 Cells The evidence that nickel compounds are human carcinogens may be considered to be unequivocal for industrial exposure to water-soluble salts (e.g., nickel chloride or sulphate) and pyrometallurgical intermediates (mostly oxides and sulphides) (12,13). The relative carcinogenic potential of different nickel compounds or species has been extensively debated. In a recent publication (14), we have shown in culture using AS52 Chinese hamster ovary cells in a colonyforming assay that to give 50% survival, a 75-fold range in administered dose was required for three water-soluble salts (chloride, sulphate, acetate) and eight relatively insoluble particulate compounds (carbonate, hydroxide, oxides and sulphides). This compared to a 10-fold range in measured cytoplasmic and nuclear "dissolved" nickel concentrations in the same cells. The particulate compounds, taken up primarily by phagocytosis, raised the nickel levels in both intracellular compartments, while the water-soluble compounds preferentially increased the cytoplasmic levels. Most of the nickel compounds tested were


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shown to be weakly mutagenic when tested for mutations at the guaninehypoxanthine phosphoribosyl transferase (gpt) gene. Some of the nickel-induced mutations have recently been characterized after clonal expansion of mutant cells and DNA amplification by the polymerase chain reaction (75). When compared to spontaneous or ethylmethane sulphonate (EMS) induced mutations, those generated by exposure to nickel compounds exhibited an increase in gene deletions relative to point mutations; the extent of which was compound specific: Ni(S0 ) > Ni(OH) > Ni S . These results clearly suggest that more than a single genotoxicological mechanism is involved in the mutagenic activity of nickel compounds. Differences in intracellular compartmentalization are believed to be important. 4



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CONCLUDING REMARKS A genetic-environmental model is the best etiological paradigm for AD (6). Multi-gene loci and a number of environmental factors appear to be involved. Linkages to loci on chromosome 1,14 and 21 (early onset AD), and chromosome 19 (late onset) have been established; the apoE gene is located on the latter. Although more AD patients need to be studied, the apoE4 genotyping results reported here do not suggest an inherited susceptibility involving this locus with respect to the GI absorption of aluminum. Nevertheless, the identification of a significant proportion of high absorbers may well have toxicological implications since aluminum is known to be neurotoxic. The 10-fold variation observed in basal and induced MT levels, as well as the 2.7-fold difference in induction capacity, likely reflect both environmental and experimental factors, and possibly genetic (inter-individual) differences. Since the variations in induction capacities were comparable in smokers and non-smokers, and a relationship between constitutive MT levels and number of cigarettes smoked was not evident, it appears that smoking was not a significant environmental factor in the young people examined. [On average, they smoked 12 ± 7 cigarettes per day; 30 was the highest]. Presumably, the exposure to Cd via tobacco smoke did not alter the effective cadmium concentration enough to affect the basal MT levels in the mononuclear cells. This is an important observation and facilitates the application of the biological monitoring techniques described in assessing environmental exposures to cadmium and mercury or perhaps in an evaluation of inherent susceptibilities/protection of exposed individuals to/from toxic effects. Reduced fertility reported in female dental assistants exposed to mercury vapour (16) might be a pertinent example. The mutant profile analysis reviewed suggests that induction of mutations at levels only slightly above background may indeed be indicative of the mutagenic potential of nickel compounds. Molecular biology techniques can thus serve an important complementary role to more traditional mutagenicity testing. The


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results reported also illustrate that mutagenic profiles may well be compound specific. Future studies are needed to assess whether the experimental approach and findings described can be extended to assessments of compound-specific acquired genetic damage in humans.


ACKNOWLEDGMENTS The following colleagues participated in the study describing GI absorption of aluminum in healthy volunteers and Alzheimer's patients: J.S. Reiach (Honours Biology and Pharmacology Program, Department of Biology), and B.L. Gibson, M.D. (Department of Clinical Epidemiology and Biostatistics), McMaster University; D.W. Molloy, M.D. and D. Strang, M.D., Geriatric Research Group, Hamilton Civic Hospitals, Henderson General Division, Hamilton, Ont; and D. Gauvreau, Ph.D. and C. Bétard, Ph.D., Project IMAGE/Recherche Alzheimer, Centre Hospitalier Côte-des-Neiges, Montreal, Que. My co-investigators for the metallothionein induction work were: C.B. Harley, Ph.D. (Department of Biochemistry), P.S. Bhoi (Honours Biochemistry Program) and G.P. Doherty (Arts and Science Program), McMaster University. Co-workers in the characterization of nickel-induced mutations in AS52 cells were: F.E. Rossetto, Ph.D. and G.G. Fletcher, M.Sc. (Department of Biochemistry) and J.D. Turnbull, Ph.D., M.D. (Department of Medicine), McMaster University. Financial assistance for the work described is gratefully acknowledged from a number of government agencies (NSERC, MRC, and the Ontario Workplace Health and Safety Agency), McMaster University (EcoResearch Chair's Program in Environmental Health) and industry (The Nickel Producers Environmental Research Association, Durham, N.C., and the International Lead Zinc Research Organization, Research Triangle Park, N.C.). LITERATURE CITED 1. 2. 3. 4. 5. 6. 7.

Nieboer, E.; Rossetto, F.E.; Turnbull, J.D. Toxicol. Lett. 1992; 64/65, 25-32. Office of Technology Assessment. Summary, Genetic Monitoring and Screening in the Workplace, Report OTA-BA-456, Washington: Congress of the United States, 1990. Calabrese, E.J. J. Occup. Med. 1986; 28, 1096-1102. Perera, F.P. JNCI 1986; 78, 887-898. Federation of American Societies of Experimental Biology. FASEB J. 1991; 5(1), 1-78. Nieboer, E.; Gibson, B.L.; Oxman, A.D.; Kramer, J.R. Environ. Rev. 1995; 3, 29-81. Corder, E.H.; Saunders, A.M.; Strittmatter, W.J.; Schmechel, D.E.; Gaskell, P.C.; Small, G.W.; Roses, A.D.; Haines, J.L.; Pericak-Vance, M.A. Science 1993; 261, 921-923.


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8. Corder, E.H.; Saunders, A.M.; Risch, N.J.; Strittmatter, W.J.; Schemechel, D.E.; Gaskell, Jr. P.C.; Rimmler, J.B.; Locke, P.A.; Conneally, P.M.; Schmader, K.E.; Small, G.W.; Roses, A.D.; Haines, J.L.; Pericak-Vance, M.A. Nature Genet. 1994; 7, 180-184.

Harley, C.B.; Menon, C.R.; Rachubinski, R.A.; Nieboer, E. Biochem. J. 1989; 262, 873-879. 10. DaSilva, J.J.R.F.; Williams, R.J.P. The Biological Chemistry of the Elements, Clarendon Press, Oxford, UK, 1991. 11. Pattanaik, Α.; Shaw, IIIC.F.;Petering, D.H.; Garvey, J.; Kraker, A.J. J. Downloaded by STANFORD UNIV GREEN LIBR on October 1, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0654.ch016

9.

12.

Inorg. Biochem. 1994; 54, 91-105. Doll, R. Scand. J.Work Environ. Health 1990; 16, 1-82.

13. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol. 49, Chromium, Nickel and Welding. International Agency for Research on Cancer, Lyon, France, 1990. 14. Fletcher, G.G.; Rossetto, F.E.; Turnbull, J.D.; Nieboer, E. Environ. Health Perspect. 1994; 102 (Suppl. 3), 69-79. 15. Rossetto, F.E.; Turnbull, J.D.; Nieboer, E. Sci. Total Environ. 1994; 148, 201-206. 16. Rowland, A.S.; Baird, D.D.; Weinberg, C.R.; Shore, P.L.; Shy, C.M.; Wilcox, A.J. Occup. Environ. Med. 1994; 51, 28-34.


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