Faculty Bibliography

Participants at the Napa Conference on Genetic and Molecular Ecotoxicology assessed the status of this field in light of heightened concerns about the genetic effects of exposure to hazardous substances and recent advancements in our capabilities to measure those effects. We present here a synthesis of the ideas discussed throughout the conference, including definitions of important concepts in the field and critical research needs and opportunities. While there were many opinions expressed on these topics, there was general agreement that there are substantive new opportunities to improve the impact of genetic and molecular ecotoxicology on prediction of sublethal effects of exposure to hazardous substances. Future studies should emphasize integration of genetic ecotoxicology, ecological genetics, and molecular biology and should be directed toward improving our understanding of the ecological implications of genotoxic responses. Ecological implications may be assessed at either the population or ecosystem level; however, a population-level focus may be most pragmatic. Recent technical advancements in measuring genetic and molecular responses to toxicant exposure will spur rapid progress. These new techniques have considerable promise for increasing our understanding of both mechanisms of toxicity on genes or gene products and the relevance of detrimental effects to individual fitness

Recent studies have examined the relationship between genetic polymorphisms of the human cytochrome P-4501A1 (CYP1A1) gene and lung cancer susceptibility. We have quantified genotypic frequencies and measured gene expression in the CYP1A1 gene within racially diverse groups in order to determine the relationship between genotype and transcriptional regulation of the CYP1A1 gene. Lymphocytes were obtained from 68 individuals of European-American, African-American, and Asian descent, and CYP1A1 gene inducibility was measured in mitogen-stimulated cells. CYP1A1 gene inducibility was significantly lower in African-Americans than in European-Americans or Asians, while several other population parameters were found to have no effect on gene expression levels. Restriction fragment length polymorphism analysis of lymphocyte DNA following MspI restriction enzyme digestion revealed a significant difference in the frequencies of CYP1A1 genotypes between European-Americans and Asians. The only homozygous variants detected were of Asian descent. The frequencies of CYP1A1 genotypes in all races conformed to Hardy-Weinberg genotypic equilibrium. When CYP1A1 gene inducibility was compared to CYP1A1 genotype, no significant correlations were found. These studies, along with our previous survey of CYP1A1 gene expression in creosote-exposed workers, add further support to the use of CYP1A1 gene inducibility as a potential marker of polycyclic aromatic hydrocarbon exposure in human populations

Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA) was used to characterize stocks of striped bass Morone saxatilis and to estimate their relative contributions during the fall of 1989 to the mixed coastal fishery at eastern Long Island, New York. Mitochondrial DNA was obtained from reference samples of striped bass collected during the spring of 1989 from the Hudson River, New York, and four spawning areas of the Chesapeake Bay (Choptank, Rappahannock, and Potomac rivers and the upper Chesapeake Bay). Five mtDNA major length genotypes were detected in these fish, and significant differences in their frequencies were observed between the Hudson River and Chesapeake Bay samples. An mtDNA minor length genotype found in some fish (13%) from the Chesapeake Bay and absent from all Hudson River samples provided a second discriminatory character. By using a constrained generalized least squares approach, we estimated that the Hudson River and Chesapeake Bay stocks contributed 73% (95% confidence interval 50-87%) and 27% (95% confidence interval 13-51%) respectively, of the mixed fishery sample. The probability of the Hudson River contribution exceeding the Chesapeake Bay contribution in this sample was more than 95%. These results suggest that the Hudson River contribution to the mixed coastal fishery was greater in 1989 than reported in earlier studies. We also found no differences in mtDNA major length genotype frequencies among as many as 11 year-classes within the Hudson River, Chesapeake Bay, or Roanoke River spawning systems. These results indicate that mtDNA RFLP genotypes in these striped bass stocks are temporally stable within a fisheries context. An advantage to mtDNA analysis over phenotypic approaches is that, because mtDNA genotype frequencies are not subject to environmentally induced variation, efforts subsequent to an initial survey can focus on characterizing the mixed coastal fishery

Mitochondrial DNA (mtDNA) was analyzed to determine the genetic relatedness of striped bass (Morone saxatilis) populations in tributaries to the Gulf of St. Lawrence and the Bay of Funday. Mitochondrial DNA genotype frequencies were determined for striped bass from the Shubenacadie River (Bay of Fundy) and the Miramichi and Tabusintac rivers (Gulf of St. Lawrence). These mtDNA genotype frequencies were compared with those of striped bass representative of the Atlantic coastal migratory stock originating in the Hudson River and Chesapeake Bay. Differences in the frequencies of mtDNA length variants permitted discrimination of the Shubenacadie River from the Miramichi River and Tabusintac River populations and all three Canadian populations from the U.S. spawned coastal migratory stock. No difference in the frequency of mtDNA length variants was observed between Tabusintac River and Miramichi River striped bass. Heteroplasmy for mtDNA length variants was observed in 35% of Gulf of St. Lawrence fish, the highest frequency observed in any striped bass population. These results highlight the genetic heterogeneity of these Canadian striped bass populations and their distinctiveness from U.S. stocks. Future efforts to restore these depleted Canadian striped bass populations should consider the impact of stock transfer on endemic striped bass gene pools

Levels of cytochrome P4501A mRNA were measured in Atlantic tomcod caged in the effluents of a bleached kraft pulp and paper mill on the Miramichi River, NB, Canada and two sites downstream, and compared to levels in fish caged upstream of the mill and in unexposed laboratory-maintained tomcod. Additionally, the rate of clearance of CYP1A mRNA in tomcod exposed at the mill and transferred to clean water was compared to that observed in tomcod treated in the laboratory with a single dose of 2, 3, 7, 8-TCDD (0.5 mu g/kg). Levels of CYP1A mRNA in tomcod caged upriver of the mill were low and comparable to those observed in untreated laboratory control fish. Levels of CYP1A mRNA were elevated up to 11-fold in tomcod caged at the mill site and a gradient in levels of gene expression was seen at the two downstream sites. Tomcod i.p. injected with a single dose of 2, 3, 7, 8-TCDD exhibited a profile of prolonged CYP1A mRNA induction of at least 25 days. Levels of CYP1A mRNA in tomcod caged at the mill site and transferred to clean water remained significantly induced for at least 3 days post-transfer. In combination, these results suggest that CYP1A mRNA was induced in caged tomcod by constituents of the mill's effluents and that the rates of clearance of CYP1A mRNA may provide additional information concerning the identity of environmental inducers

We tested the ability of cellular oncogene (c-onc) probes to identify F1 hybrids and the lineage of known backcrosses within the fish genus Morone. Total DNA was isolated from five to 14 individuals per North American Morone species (striped bass, white bass, white perch, and yellow bass). The DNA was digested with two restriction enzymes, Eco RI and Hin dIII, Southern blotted, and hybridized to six different c-onc probes including v-abl, v-erb B, c-myc, c-H-ras, c-K-ras, and v-src. We found fixed genotypic differences among the four species for all six probes in single restriction enzyme digests. The heritability of these nuclear DNA genotypes was evaluated in hatchery-produced F1 Morone hybrids (striped bass x white bass and striped bass x white perch) tested with the six informative single probe/restriction enzyme combinations. All F1 individuals exhibited heterozygosity in all diagnostic nuclear DNA fragments, confirming the Mendelian inheritance of these genotypes in these fish. Furthermore, analysis of these nuclear DNA genotypes in hatchery-produced backcrosses of F1 hybrids striped bass x (white bass x striped bass) detected both recombinant and parental genotypes at all six polymorphic c-onc sequences. The lineage of suspected Morone hybrids of unknown descent collected from Lewis Smith Lake, Alabama, and from the Occoquan River, Virginia, was determined using the c-onc probes. Our results suggest that c-onc probes are suitable markers to unequivocally identify F1 hybrids and backcrosses and to quantify introgression in natural populations of fishes. The addition of RFLP analysis of mtDNA provided a complete ancestral history of individual fish

Levels of P4501A mRNA in tomcod collected from the Miramichi River, Canda and the Hudson River, New York were measured by slot blot analyses after the fish had been depurated and ip. injected with Aroclor 1254, PCB congener 77, or PCB congener 105. Elevated expression of P4501A mRNA was observed in congener 77-treated Miramichi tomcod, whereas no induction was seen in the Hudson River fish. Tom cod from both rivers were induced by injection with beta-naphthoflavone. These results suggest that the prior exposure history of the Hudson River tomcod may have affected their non-inducibility by treatment with PCB congener 77

We sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, we found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached by 5 days. Intraperitoneal injection of beta-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers

We used restriction endonuclease analysis of mitochondrial DNA (mtDNA) to differentiate among spawning stocks of American shad (Alosa sapidissima). Highly purified mtDNA was isolated from shad from four major spawning rivers: the St. John's (Florida), the Delaware, and the Hudson in the United States and the Miramichi in New Brunswick, Canada. Primarily four- and-five-base-cutting restriction enzymes were used to prepare both individual enzyme profiles and composite genotypes. Three separate spawning stocks, St. John's, Delaware-Hudson, and Miramichi, could be distinguished based on frequency differences in mtDNA genotypes generated by single restriction enzyme digests. We could not distinguish Delaware from Hudson River shad. Only a single definitive restriction site polymorphism was observed among all samples, but polyacrylamide gel electrophoretic mobility variants were common. Eco RI, Dde I, and Rsa I revealed stock-specific mtDNA genotypes. The frequencies of some genotypes occurred in latitudinal clines. Fifty-seven of 81 fish showed individual-specific composite genotypes. Geographic partitioning of genotypes suggests that mtDNA analysis may be useful for the identification of some American shad stocks and their relative contributions to mixed coastal fisheries