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.

Responses of fish populations and communities to pulp mill effluents: a holistic assessment.

Fish populations residing in a river receiving bleached kraft mill effluents (BKME) and in an uncontaminated river were investigated to evaluate causal relationships between exposure to BKME and various indicators of fish health. The Index of Biotic Integrity demonstrated that species richness and composition were much lower in the contaminated river with an obvious imbalance in the trophic structure of the fish community. Biomolecular and biochemical responses such as DNA damage and elevated activity of detoxification enzymes indicated that fish in the contaminated river had been exposed to toxicants. The status of various condition indices such as the liver and visceral somatic indices suggested metabolic and nutritional imbalances in sunfish as a result of exposure to pulp mill effluents. Fish populations in the contaminated river also demonstrated an abnormal size distribution and age structure. Female redbreast sunfish from the BKME-impacted river contained a large number of atretic oocytes and had lower serum levels of estradiol than fish from the reference site. These observations plus data from previous developmental toxicity studies suggest that the primary mechanism by which BKME may affect fish populations in this river is through reproductive dysfunction and recruitment failure. Decreased recruitment may have caused a reduction in population size and resulted in decreased competition and increased resource availability. With increased food and habitat availability, more energy may have been available for growth and lipid storage of survivors in the contaminated river.

Biological markers of environmental and ecological contamination: an overview.

An approach, using biomarkers (biological responses) for assessing the biological and ecological significance of contaminants present in the environment is described. Living organisms integrate exposure to contaminants in their environment and respond in some measurable and predictable way. Responses are observed at several levels of biological organization from the biomolecular level, where pollutants can cause damage to critical cellular macromolecules and elicit defensive strategies such as detoxication and repair mechanisms, to the organismal level, where severe disturbances are manifested as impairment in growth, reproduction, developmental abnormalities, or decreased survival. Biomarkers can provide not only evidence of exposure to a broad spectrum of anthropogenic chemicals, but also a temporally integrated measure of bioavailable contaminant levels. A suite of biomarkers are evaluated over time to determine the magnitude of the problem and possible consequences. Relationships between biomarker response and adverse ecological effects are determined from estimates of animal health and population structure.

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.

Sequential expression of biomarkers in Bluegill Sunfish exposed to contaminated sediment.

The temporal expression of various biological rsponses was determined in Bluegill SunfishLepomis macrochirus exposed under controlled laboratory conditions to sediment containing high concentrations of polynuclear aromatic hydrocarbons, polychlorinated biphenyls and heavy metals. Liver, gill, blood, kidney, brain, spleen and intestine were removed from Sunfish sampled at 1, 2, 4, 8, 16, and 40 weeks post-exposure. Biomarker data were recorded for specific proteins, enzymatic activities, DNA integrity, and histopathology. Biomarkers in the laboratory exposed fish were similar to those of indigenous Sunfish sampled from the site of origin of the contaminated sediment. Several patterns of development of biomarkers over time were also evident. For example, the responses of certain biomarkers are not time-dependent (i.e., intestine and gill ATPase activities) while that of others, such as brain ATPase activity, liver cytochrome P450 and NADPH content, stress proteins, chromatin proteins and DNA strand breaks, fluctuate over time. Still other biomarkers, such as EROD activity, zinc protoporphyrin content of the blood, and DNA adducts, showed marked increases over time. Such patterns need to be considered when comparing laboratory and field results and deciding which biomarkers to use for biomonitoring programs. Implications for natural selection and population/community level responses are also discussed.

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.

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.

The relationship between benzo(a)pyrene diol-epoxide-DNA adducts and mutagenicity in the CHO/HPGRT assay.

We have studied the relationship between DNA adducts in Chinese hamster ovary (CHO) cells and mutagenicity as determined in the CHO/hypoxanthine-guanine phosphoribosyltransferase assay. The cells were treated with benzo(a)pyrene 7,8-diol (BP-diol) in the presence of a bioactivation system, S9 mix. DNA binding by bioactivation of BP-diol with S9 mix occurred with both stereoisomers of benzo(a)pyrene diol-epoxide (BPDE) in approximately equal amounts. The number of BPDE-DNA adducts (21-260 adducts/10(6) nucleotide base pairs) increased with increasing treatment concentrations of BP-diol (1.4-7.0 microM). A linear relationship was observed between the number of BPDE-DNA adducts and mutagenicity (89-605 mutants/10(6) cloneable cells) over the concentration range of BP-diol assayed.

An in vitro approach to studying cutaneous metabolism and disposition of topically applied xenobiotics.

The extent to which cutaneous metabolism may be involved in the penetration and fate of topically applied xenobiotics was examined by metabolically viable and structurally intact mouse skin in organ culture. Evidence that skin penetration of certain chemicals is coupled to cutaneous metabolism was based upon observations utilizing [14C]benzo[a]pyrene (BP). As judged by the recovery of radioactivity in the culture medium 24 hr after in vitro topical application of [14C]BP to the skin from both control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced C3H mice, skin penetration of BP was higher in the induced tissue. All classes of metabolites of BP were found in the culture medium; water-soluble metabolites predominated and negligible amounts of unmetabolized BP were found. As shown by enzymatic hydrolysis of the medium, TCDD induction resulted in shifting the cutaneous metabolism of BP toward the synthesis of more water-soluble conjugates. Differences in the degree of covalent binding of BP, via diol epoxide intermediates to epidermal DNA, from control and induced tissues were observed. These differences may reflect a change in the pathways of metabolism as a consequence of TCDD induction. These results indicated that topically applied BP is metabolized by the skin during its passage through the skin; and the degree of percutaneous penetration and disposition of BP was dependent upon the metabolic status of the tissue. This suggests that cutaneous metabolism may play an important role in the translocation and subsequent physiological disposition of topically applied BP.

Multiple isoacceptor forms of several transfer ribonucleic acids in a mutant yeast strain.

By use of reverse phase 5 chromatography, a strain of Saccharomyces cerevisiae (XB 109-5B) has been shown to exhibit multiple isoaccepting forms for several of the transfer ribonucleic acids (tRNAs). This is in contrast with a standard wild-type strain where only one acceptor is found for each tRNA studied. Multiple peaks for tRNATyr, tRNAPhe, tRNASer, and tRNAVal have been detected for strain XB 109-5B. However, the observation of multiple isoacceptors cannot be extended to all tRNAs in this strain since tRNAAsp appears as a single form that is the same as in the wild type. The appearance of multiple peaks was found to depend on the growth conditions of the cells. The tRNA profiles of XB 109-5B that was grown rapidly with vigorous aeration differed the most from profiles of comparably grown wild-type yeast, whereas tRNA from this mutant, grown without shaking or supplementary aeration, appeared the same as the wild type. The minor nucleoside composition of the isoacceptors of tRNAPhe was obtained.

Occurrence and Distribution of Proteinase of Streptococcus faecalis var. liquefaciens.

Shugart, Lee R. (University of Tennessee, Knoxville), and Raymond W. Beck. Occurrence and distribution of proteinase of Streptococcus faecalis var. liquefaciens. J. Bacteriol. 92:338-341. 1966.-The proteolytic enzyme produced by Streptococcus faecalis var. liquefaciens (ATCC 13398) was shown to be an exoenzyme. The production of the proteinase was followed in growing cultures, and its distribution was compared with that of the intracellular enzymes reduced nicotinamide adenine dinucleotide (NADH(2)) peroxidase and lactate dehydrogenase. The proteinase appeared in the culture medium prior to the stationary phase of growth, whereas the other enzymes could be found only in whole cells. Fractionation of whole cells by sonic treatment and by treatment with lysozyme showed the proteinase to be associated primarily with the cell wall and cell membrane, and NADH(2) peroxidase to be associated only with the cytoplasmic fractions.