DNA alterations and enzyme activities in Japanese medaka (Oryzias latipes) exposed to diethylnitrosamine.

Several molecular and biochemical markers of genotoxicity were adapted for measurement in the medaka, and were used to describe the effects of treatment of the organism with diethylnitrosamine (DEN). DEN treatment inhibited the activity of a detoxication enzyme activity (ethoxyresorufin-O-deethylase) and increased the activity of glutathione-S-transferase. This pattern of response has been described in preneoplastic rodent cells. No O6-ethyl guanine adducts were detected, and a slight, but statistically significant, increase in DNA strand breaks was observed. These results are consistent with the hypothesis that prolonged exposure to high levels of DEN induced alkyltransferase activity which enzymatically removes any O6-ethyl guanine adducts but does not result in strand breaks or hypomethylation of the DNA such as might be expected from excision repair of chemically modified DNA.

Environmental genotoxicity: probing the underlying mechanisms.

Environmental pollution is a complex issue because of the diversity of anthropogenic agents, both chemical and physical, that have been detected and catalogued. The consequences to biota from exposure to genotoxic agents present an additional problem because of the potential for these agents to produce adverse change at the cellular and organismal levels. Past studies in genetic toxicology at the Oak Ridge National Laboratory have focused on structural damage to the DNA of environmental species that may occur after exposure to genotoxic agents and the use of this information to document exposure and to monitor remediation. In an effort to predict effects at the population, community, and ecosystem levels, current studies in genetic ecotoxicology are attempting to characterize the biologic mechanisms at the gene level that regulate and limit the response of an individual organism to genotoxic factors in their environment.

Examination of adduct formation in vivo in the mouse between benzo(a)pyrene and DNA of skin and hemoglobin of red blood cells.

We are interested in devising techniques which will allow us to measure and quantitate exposure to chemical carcinogens and which eventually can be used in risk analysis with humans. Our recent research with HPLC/fluorescence has demonstrated that we can detect, identify, and quantitate the binding of benzo(a)pyrene (BaP) with DNA of mouse skin. The technique not only allows femtomole amounts of BaPDE associated with DNA isolated from a single mouse skin to be detected using conventional instrumentation, but also establishes the stereochemical origin of the adduct, and has been employed in the investigation reported here to estimate the concomitant binding of BaP to hemoglobin in vivo. The temporal existence of BaPDE/DNA adducts in mouse skin over a 5-week period showed that at 35 days after treatment, approximately 15% of the initial adducts were still detectable even though DNA turnover would predict that they should have been deleted from the genome. The concentration of the major covalently bound adduct, anti-BaPDE/deoxyguanosine, relative to the total BaPDE/DNA adduct population remained essentially constant during the 5-week period. It is known that topically applied BaP is absorbed, metabolized, and excreted by the mouse. Examination of hemoglobin of mouse RBCs 24 hr after BaP treatment revealed covalent adduct formation exclusively via anti-BaPDE. The dose response of adduct binding to hemoglobin and DNA appeared to be similar.

Molecular responses as indicators of marine pollution: DNA damage and enzyme induction in Limanda limanda and Asterias rubens.

During a survey from 26 August through 13 September 1991, specimens of the flatfish, Limanda limanda (dab), and the asteroid echinoderm Asterias rubens (seastar), were collected at sampling locations along transects radiating into the North Sea from the coastal zone of The Netherlands. In homogenates of liver tissue from male dab and the digestive gland (pyloric caeca) of female seastar, DNA damage (strand breaks) and induction of the cytochrome P450-dependent monooxygenase system (MO) were determined. Areas could be described with significantly increased percentages of strand breaks (lower integrity) both in dab and seastar. However, enhanced DNA strand breaks did not correspond with contamination gradients, expressed as concentrations of polychlorinated biphenyls (PCBs) or polyaromatic hydrocarbons. MO enzyme induction in the hepatic 13,000g fraction of male dab, measured as 7-ethoxyresorufin-O-deethylase activity, was significantly enhanced in response to low ambient temperatures. Some evidence was found for the facilitation of benzo[a]pyrene hydroxylase activity expressing the enzyme induction in the microsomal fraction of pyloric caeca of seastars, at increasing PCB concentrations. DNA integrity and enzyme induction elucidate the physiologic status and might be indicative for ambient impairment within restricted areas, and not necessarily related to the presence of anthropogenic or xenobiotic substances.

Genetic and molecular ecotoxicology: a research framework.

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.

Quantitating exposure to chemical carcinogens: in vivo alkylation of hemoglobin by benzo[a]pyrene.

Mild acid hydrolysis of globin preparations from erythrocytes of mice, previously exposed topically to benzo[a]pyrene (BaP), releases tetrols which are detectable by HPLC/fluorescence analysis. If the mouse is exposed to radiolabelled BaP, radioactivity can be found in the acid-releasable tetrols. Treatment of the globin preparations prior to acid hydrolysis with proteolytic enzymes, but not enzymes that degrade nucleic acids, followed by dialysis, reduces the amount of tetrols that can be detected. Because the procedure used for the isolation of globin preparations from mouse blood precludes the presence of non-covalently bound BaP or its cellular metabolites, it is concluded that prior to acid hydrolysis, the tetrols were covalently attached to the hemoglobin, most probably as a result of the metabolic conversion of the applied carcinogen to the chemically reactive anti-diol epoxide. There is a dose response relationship between the amount of BaP applied to the skin of the mouse and the occurrence, 24 h later, of BaP adducts to hemoglobin, while the adduct, once formed, disappears with a half-life of 6 days. The amount of anti-benzo[a]pyrene diol epoxide (anti-BaPDE) binding to DNA and hemoglobin at various doses of BaP appears to be qualitatively similar.

Adduct formation in hemoglobin of the newborn mouse exposed in utero to benzo[a]pyrene.

The administration of benzo[a]pyrene topically to pregnant mice during days 13-17 of gestation results in adduct formation in the hemoglobin of the mother and progeny. Thus, exposure to a total maternal body burden of 500 micrograms of benzo[a]pyrene during the last 5 days before delivery resulted in an average level of 6.35 (+/- 0.70 S.E.M.) pg of anti-diolepoxide metabolite covalently attached per mg of hemoglobin analyzed in the mother and 1.40 (+/- 0.23 S.E.M.) in the newborn animals. These data indicate that benzo[a]pyrene administered to the skin of the mother passed across the placental membrane, either as benzo[a]pyrene or some metabolite(s), and was present in the fetal tissue as the "ultimate" carcinogenic form (anti-diolepoxide metabolite) before binding to the hemoglobin. Concomitant adduct formation in the DNA of the skin with benzo[a]pyrene in the progeny was not observed and was probably due to the small amount of carcinogen applied to the mother. The data obtained, along with previously published results [Toxicology, 34 (1985) 211], suggest the suitability of hemoglobin as a molecular dosimeter for estimating carcinogenic risk to polycyclic aromatic hydrocarbons.

Pathology and toxicology of beluga whales from the St. Lawrence Estuary, Quebec, Canada. Past, present and future.

An indigenous population of 450-500 beluga whales (Delphinapterus leucas) inhabiting the St. Lawrence Estuary has been exposed chronically for more than 50 years to a complex mixture of industrial pollutants including organochlorinated compounds (OC), polycyclic aromatic hydrocarbons (PAH) and heavy metals. From 1983 to 1990, we have necropsied 45 well preserved carcasses out of a total of 120 beluga whales reported dead over this period. Of these 45 animals, nine were affected by 10 malignant neoplasms. Fifteen animals (33%) were affected by pneumonia. Milk production was compromised in eight of 17 mature females (41%), by inflammatory changes (seven animals) and cancer (one animal) which affected the mammary glands. Opportunistic bacteria were found in pure culture, and/or in significant amounts in at least two organs in 20 belugas (44%). The concentrations of both total PCBs and highly chlorinated PCB congeners were much higher in St. Lawrence animals than in Arctic beluga whales. OC-induced immunosuppression has been repeatedly demonstrated in a wide variety of animal species. Therefore, it is probable that the immune functions of St. Lawrence beluga whales are impaired. Benzo[a]pyrene adducts were detected in 10 of the 11 St. Lawrence beluga whales of which tissues (six livers, 10/11 brains) were analyzed by a method based on HPLC. No such adducts were found in four Arctic animals. Since benzo[alpha]pyrene is one of the most potent chemical carcinogens known to man, these compounds might be responsible for some of the cancers observed in that population. Overall, our findings contrast vividly with those of others who found that cancers are exceedingly rare in free-ranging odontocete populations and that the major causes for mortalities in these populations are bacteria, parasites, and trauma.

Pathology of stranded beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary, Québec, Canada.

From June 1983 to May 1986, thirteen carcasses of stranded beluga whales from a polluted area of the St. Lawrence River, Canada were necropsied. High performance liquid chromatography was performed on the brains of three other animals to determine concentrations of benzo a pyrene (BaP). Two juvenile animals had severe multisystemic lesions one of which, a severe necrotizing dermatitis, was associated with a Herpesvirus-like particle. Four adults had five varieties of tumours. An adult had a systemic nocardiosis and a juvenile was affected ty a non 0:1 Vibrio cholerae septicemia. High concentrations of BaP adducts were found in the brains which were analyzed. Occurrence of BaP adducts in the brain of three whales of this population coincides with the high incidence of tumours. This and the previous finding of high concentrations of organochlorine in the tissues of these animals suggest an important role of industrial contaminants in the recent decrease of this population.

Characterizing biological variability in livestock blood cholinesterase activity for biomonitoring organophosphate nerve agent exposure.

A biomonitoring protocol, using blood cholinesterase (ChE) activity in livestock as a monitor of potential organophosphate nerve agent exposure during the planned destruction of US unitary chemical warfare agent stockpiles, is described. The experimental design included analysis of blood ChE activity in individual healthy sheep, horses, and dairy and beef cattle during a 10- to 12-month period. Castrated and sexually intact males, pregnant and lactating females, and adult and immature animals were examined through at least one reproductive cycle. The same animals were used throughout the period of observation and were not exposed to ChE-inhibiting organophosphate or carbamate compounds. A framework for an effective biomonitoring protocol within a monitoring area includes establishing individual baseline blood ChE activity for a sentinel group of 6 animals on the bases of blood samples collected over a 6-month period, monthly collection of blood samples for ChE-activity determination during monitoring, and selection of adult animals as sentinels. Exposure to ChE-inhibiting compounds would be suspected when all blood ChE activity of all animals within the sentinel group are decreased greater than 20% from their own baseline value. Sentinel species selection is primarily a logistical and operational concern; however, sheep appear to be the species of choice because within-individual baseline ChE activity and among age and gender group ChE activity in sheep had the least variability, compared with data from other species. This protocol provides an effective and efficient means for detecting abnormal depressions in blood ChE activity in livestock and can serve as a valuable indicator of the extent of actual plume movement and/or deposition in the event of organophosphate nerve agent release.

Quantifying adductive modification of hemoglobin from mice exposed to benzo[a]pyrene.

The present work describes a method for the detection of minute amounts of benzo[a]pyrene, as the diolepoxide metabolite, bound covalently to the hemoglobin of erythrocytes isolated from mice previously exposed to the carcinogen. The technique consists of the acid-induced removal of the pyrenyl moiety from the hemoglobin as the strongly fluorescent free tetrols and their isolation by bonded-phase extraction methods and subsequent quantitation by fluorescence/HPLC. With this procedure as little as 5 pg of tetrol can be detected. The assay was used to determine the amount of benzo[a]pyrene-hemoglobin adduct formation in mice bearing a carcinogen-induced fibrosarcoma.

Kinetic studies of Escherichia coli transfer RNA (uracil-5-)-methyltransferase.

The kinetic mechanism of a semipurified tRNA (uracil-5-)-methyltransferase (EC 2.1.1.35) preparation obtained from Escherichia coli has been studied at pH 9.0 in the presence and absence of products. The initial velocity and product inhibition patterns are consistent with a random order of addition of adenosylmethionine and transfer RNA to separate and independent binding sites on the enzyme. Values have been determined for the Michaelis and product inhibitor constants.

Selective methylation: an incorrect hypothesis.

"Selective methylation," a hypothesis proposed to explain the discrepancy found in the degree of methyl deficiency of transfer ribonucleic acid, cannot be explained on the basis of some biological phenomenon.