K-ras oncogene DNA sequences in pink salmon in streams impacted by the Exxon Valdez oil spill: no evidence of oil-induced heritable mutations

It was hypothesized in previous studies that the Exxon Valdez oil spill in Prince William Sound, Alaska, induced heritable mutations and resulted in mortality of pink salmon (Oncorhynchus gorbuscha) embryos. In one of these studies, laboratory exposure of pink salmon embryos to crude oil resulted in apparent mutation-induction in exon 1 and exon 2 of the K-ras oncogene, but no fish from the area impacted by the oil spill were analyzed. We assessed K-ras exon 1 and exon 2 DNA sequences in pink salmon from five streams that were oiled and five streams that were not oiled by the Exxon Valdez oil spill in Prince William Sound, and two streams with natural oil seeps and one stream without seeps on the Alaska Peninsula. Of the 79 fish analyzed for exon 1 and the 89 fish analyzed for exon 2, none had the nucleotide substitutions representing the mutations induced in the laboratory study. Other variable nucleotides occurred in similar proportions in oiled and non-oiled streams and probably represent natural allelic variation. These data do not support the hypothesis that heritable mutations in the K-ras gene were induced by the Exxon Valdez oil spill or oil seeps.     

Induction of Fish Vitellogenin and Alterations in Testicular Structure: Preliminary Results of Estrogenic Effects in Chub (Leuciscus cephalus)

Laboratory and field studies were carried out in order to assess, in a common cyprinid freshwater fish, the chub (Leuciscus cephalus), the feasibility of biological methods to detect and quantify estrogenic effects. In the laboratory the effects of 17beta estradiol were investigated on Vitellogenin (Vtg) induction and on testis organisation. Vtg was quantified by immunodetection using an established ELISA for carp (Cyprinus carpio) Vtg. Estradiol exposure resulted in a significant and rapid increase in plasma Vtg in both male and female chub, indicating that vitellogenic response in the chub is sensitive to estrogen(s). Histological examination of the testis in males exposed to estradiol also showed effects on the testis. Preliminary field studies on wild chub showed that fish living in a polluted river had elevated concentrations of plasma Vtg and had some alterations in the testis compared with wild fish from a reference site. Together these preliminary laboratory and field results show that chub could be an interesting fish species for further studies of biological effects of environmental estrogens in the aquatic environment.     

High genetic diversity in wild culinary-medicinal wood ear mushroom, Auricularia polytricha (Mont.) Sacc., in tropical China revealed by ISSR analysis

The genetic diversity of 40 wild Auricularia polytricha strains within ten natural populations in tropical China and five cultivated strains were analyzed by using twelve inter-simple sequence repeat (ISSR) primers. At the species level, the percentage of polymorphic loci (P) = 99.8%, the effective number of alleles (Ne) = 1.3569, Nei's gene diversity (H) = 0.2398, Shannon information index (I) = 0.3896, and total genetic diversity (Ht)= 0.2346 indicate a high level of genetic diversity in wild A. polytricha. At the population level of the wild fungus, P = 43.51%, Ne = 1.2675, H = 0.1702, I = 0.2504. However, the genetic diversity of cultivated strains was the lowest of all populations (Ne = 1.1584, H = 0.0940, I = 0.1440). A moderate degree of genetic differentiation (G(st) = 0.347) among the sampled wild populations was detected based on Nei's gene diversity analysis, suggesting that 65.3% of variation existed within this population. The high genetic variation level within wild populations may mostly result from a low level of gene flow (N = 0.9408) and random genetic drift.     

Mechanisms of in-vitro-selected daptomycin-non-susceptibility in Staphylococcus aureus

Daptomycin is highly active against Staphylococcus aureus, including multidrug-resistant strains and those with reduced susceptibility to vancomycin. However, daptomycin-non-susceptible (Dap^NS) strains [minimum inhibitory concentration (MIC) >1 mg/L] have been derived clinically and in vitro. The mechanism(s) by which this occurs is incompletely understood, but existing data indicate that it is multifactorial. Dap^NS derivatives of one laboratory and three clinical strains of S. aureus produced using gradient plates were evaluated. The Dap^NS phenotype included increases in glycopeptide and nisin MICs and in some instances defective autolysis and reduced susceptibility to lysostaphin lysis. Amino acid substitutions in MprF, YycG (WalK), or both, were identified in all Dap^NS strains. Reduced cytochrome c binding and ability of daptomycin to depolarise whole cells correlated with the Dap^NS phenotype, consistent with an increase in cell surface positivity. Gene expression data revealed increased expression of vraS, one member of a two-component system involved in the regulation of cell wall biosynthesis, in three of five Dap^NS strains. The pathway to the Dap^NS phenotype is not linear, as variable genetic and phenotypic changes may result in identical increases in MICs.     

Variation in blood cell DNA in Carassius carassius from ponds near Chernobyl, Ukraine

Blood from approximately 400 crucian carp, Carassius carassius, from 12 ponds in the Ukraine, was analysed by flow cytometry to assess the possible relationships between chronic contaminant exposure and variation in the cellular DNA content. The ponds were located 20--30 km from Chernobyl in areas that received 3.7 × 1010--3.7 × 1011 Bq 137Cs km-2 after the 1986 nuclear accident, as well as other radioactive and chemical contaminants. The fish populations consisted of both diploid and triploid individuals and the ploidy varied between ponds. Analysis of whole blood revealed aneuploid-like patterns in the DNA histograms of some fish, as well as hyperdiploid shoulders of the G0/G1 peak. The coefficient of variation (CV) of the G0/G1 peak has previously been employed to assess the variability in the DNA content of cells within individuals. The CV of individual fish varied between and within locations and very large CVs were found for some individuals. In some fish, DNA histograms showed a typical diploid or triploid cell population together with a smaller haploid population. Variations in the cellular DNA content similar to those reported here have been associated with exposure to radiation and other genotoxic agents in laboratory and field studies. However, the abnormalities we observed were not correlated with known contaminant distributions. While further work is needed, particuarly in areas with substantially higher levels of radioactivity, these results suggest that the Chernobyl accident may have long-term genetic consequences for wild organisms inhabiting contaminated areas     

Toxicological pathology in laboratory fish: an evaluation with two species and various environmental contaminants

This paper presents an overview of the studies carried out in small laboratory fish species to investigate the usefulness of histopathology as a tool in aquatic toxicology. The studies were performed with medaka (Oryzias latipes) and guppy (Poecilia reticulata) that were exposed to a variety of environmental contaminants for one and three months. Besides the measurement of routine toxicological parameters, total body histopathology was carried out. The data are summarized and evaluated in view of their contribution to the knowledge of toxicology, the specific responsiveness of animals and tissues, and the application in ecotoxicology. It is concluded that histopathology of (small) fish exposed to environmental contaminants may provide useful information as to target organs and mechanism of action: moreover, this technique can be more sensitive than routine parameters.     

Effects of benzo[a]pyrene on mitochondrial and nuclear DNA damage in Atlantic killifish (Fundulus heteroclitus) from a creosote-contaminated and reference site

Benzo[a]pyrene (BaP) is a known genotoxicant that affects both mitochondrial and nuclear DNA (mtDNA, nDNA). Here, we examined mtDNA and nDNA damage in the Atlantic killifish (Fundulus heteroclitus) from a highly contaminated Superfund site (Elizabeth River, VA, USA) and from a reference site (King's Creek, VA, USA) that were dosed with 10 mg/kg BaP. Using the long amplicon quantitative PCR technique, we observed similar increases in mitochondrial and nuclear DNA damage in King's Creek fish treated with BaP. Killifish from the Elizabeth River showed high levels of basal nDNA and mtDNA damage compared to fish from the reference site, but the level of damage induced due to BaP treatment was much lower in Elizabeth River killifish compared to King's Creek fish. Laboratory-reared offspring from both populations showed increased BaP-induced damage in mtDNA, relative to nDNA. Similar to the adult experiment, the Elizabeth River larvae had higher levels of basal DNA damage than those from the reference site, but were less impacted by BaP exposure. Measurements of oxidative DNA damage (8-oxo-deoxyguanine by LC–MS/MS) showed no differences among treatment groups, suggesting that the majority of DNA damage is from covalent binding of BaP metabolites to DNA. This study shows for the first time that mitochondria can be an important target of BaP toxicity in fish, indicating that BaP exposures could have important energetic consequences. Results also suggest that multi-generational exposures in the wild may lead to adaptations that dampen DNA damage arising from BaP exposure.     

Sublethal Identification of Susceptible Individuals: Using Swim Performance to Identify Susceptible Fish While Keeping them Alive

One branch of ecotoxicology has focused on identifying genetic markers in fish that are associated with susceptibility to toxic compounds. In laboratory studies, a common approach has been to compare the genetic variation in fish that die first in time-to-death studies to that found in fish that live longer or survive the exposure. Studies of this kind would benefit from the ability to identify living individuals as susceptible, as these individuals could then be used to answer currently unanswerable questions. The purpose of this mini-review is to suggest that post-exposure swim performance can be used to sublethally discriminate between susceptible and resistant individual fish after these fish have been exposed to environmental stressors, particularly heavy metals.     

Identification of Bacteria Isolated from an Oligotrophic Lake with Pesticide Removal Capacities

We studied the growth and capacities for pesticides removal of bacterial strains isolated from the Laguna Grande, an oligotrophic lake at the South of Spain (Archidona, Málaga). Strains were isolated from water samples amended with 10 and 50 g/ml of nine pesticides: organochlorinated insecticides (aldrin and lindane), organophosphorous insecticides (dimetoate, methyl-parathion and methidation), s-triazine herbicides (simazine and atrazine), fungicide (captan) and diflubenzuron (1-(-4-chlorophenyl)-3-(2,6-difluorobenzoyl urea), a chitinase inhibitor. The majority of the strains belonged to the genera Pseudomonas and Aeromonas and only 9% of the total of strains were Gram positive. From all the strains isolated, only 22 showed a wide growth range in all the pesticides tested and 4 of them were chosen for pesticide removal studies. The genetic identification of these strains showed their affiliation to Pseudomonas pseudoalcaligenes, Micrococcus luteus, Bacillus sp. and Exiguobacterium aurantiacum. These last two strains were those that showed the highest pesticide removal capacities and a high bacterial growth.     

Survey of <Emphasis Type="Italic">Ephedra </Emphasis>resources in the Northern Areas of Pakistan and their genetic diversity

More than 400 species of medicinal plants grow in the Northern Areas of Pakistan, including Ephedra plants. To investigate the wild Ephedra plant resources in the area, we surveyed the medicinal plants and collected 71 specimens from 18 collecting sites to analyze their genetic variation. The DNA sequences of the internal transcribed spacers 1 and 2 (ITS 1 and 2) of nuclear ribosomal DNA and a noncoding sequence of chloroplast DNA (trn L/F) were analyzed. This DNA data analysis and external morphological features were used to confirm the species of the specimens, and it was found that E. intermedia was the major species in the area and that E. gerardiana and E. przewalskii were present sporadically. Although it inhabits a relatively small area in comparison with the northwestern Chinese provinces, the DNA sequence of E. intermedia in the Northern Areas of Pakistan was significantly more heterogeneous than the same species grown in those neighboring regions. Most of the E. intermedia specimens contained more than 0.7% ephedrine alkaloids, fulfilling the requirement of the Japanese Pharmacopoeia; thus, the Ephedra plants in the area are a genetic and medicinal resource of great importance.     

乙型脑炎减毒活疫苗病毒群体的异质性分析

 目的  通过观察疫苗病毒群体中病毒个体的遗传和生物学特征,分析SA14-14-2株乙型脑炎减毒活疫苗病毒的均一性,为疫苗病毒的遗传稳定性提供实验证据。 方法   对3批疫苗病毒连续进行3次蚀斑纯化后,各挑选8个蚀斑纯化株,扩大培养一代。比较24个病毒纯化株的包膜（envelope,E）蛋白基因序列、单斑培养病毒滴度以及蚀斑特征。 结果   24个纯化株中共有6个病毒株（25%）的E蛋白核苷酸发生变化,其中2株（8.3%）的核苷酸变异导致其编码氨基酸发生改变,这些变异占总核苷酸变异数的28.6%。动物实验表明,这2个纯化株没有引起小鼠神经毒力变化。在所有24个纯化株中,我国2010年版药典规定的影响疫苗病毒遗传稳定性的8个E蛋白关键位点的氨基酸均未发生改变。24个纯化株病毒的蚀斑大小和形态以及单斑培养滴度均无明显差异。 结论   疫苗病毒具有典型的准种群体多样性特征,但在群体病毒中未检测到E蛋白氨基酸位点为野生型病毒位点的个体病毒,因此,疫苗病毒具有很好的减毒群体特征和遗传稳定性。     

Cytochrome b sequences in black-crowned night-herons (Nycticorax nycticorax) from heronries exposed to genotoxic contaminants

DNA sequence analysis of a 215 base-pair region of the mitochondrial cytochrome b gene was used to examine genetic variation and search for evidence of an increased mutation rate in black-crowned night-herons. We examined five populations exposed to environmental contamination (primarily PAHs and PCBs) and one reference population from the eastern U.S. There was no evidence of a high mutation rate even within populations previously shown to exhibit increased variation in DNA content among somatic cells as a result of petroleum exposure. Three haplotypes were observed among 99 individuals. The low level of variability could be evidence for a genetic bottleneck, or that cytochrome b is too conservative for use in population genetic studies of this species. With the exception of one population from Louisiana, pair-wise Phi_st estimates were very low, indicative of little population structure and potentially high rates of effective migration among populations.     

Genetic analysis of the behavioral response to d-amphetamine in mice

The ambulatory and rearing responses to d-amphetamine were studied in a battery of recombinant inbred strains and in three closely related strains: C57BL/6J, C57BL/10J, and C57BL/6By. Differences in the increase of ambulation (stimulation) caused by d-amphetamine were seen between C57BL/6By and the other two C57BL strains. Analysis of F₁ and backcross matings suggests a one-gene model. A mutation at the genetic locus that affects the response to d-amphetamine seems to have taken place in the C57BL/6By strain. Strain differences in the decrease of rearing behavior (inhibition) produced by the drug were observed in recombinant inbred strains. Although the genetic analysis is not conclusive, it appears to be compatible with regulation by a single major gene.The two single-gene models reported here (one affecting the stimulatory response and the other the inhibitory response to d-amphetamine) may be useful in the study of neural mechanisms involved in stimulation and inhibition of behavior by d-amphetamine.     

Impact of season on liver mitochondrial oxidative stress and the expression of HSP70 in grey mullets from contaminated estuary

Heat shock proteins (HSPs) are the ubiquitous feature of cells in which these proteins cope with stress induced denaturation of other proteins. HSP70 is found to play a primary role in cellular defense under stress condition. In the present investigation, the seasonal impact on environmental stress induced mitochondrial HSP70 (mtHSP70) expression in the liver mitochondria was examined in grey mullets, Mugil cephalus living in the Ennore estuary (polluted site) was compared with the Kovalam estuary (unpolluted site) over the course of two seasons viz monsoon and summer from April 2006 to March 2008. Oxidative stress was determined along with mtHSP70 expression studies in fish liver mitochondria collected from these two estuaries for both the seasons. The liver mitochondria of grey mullet fish collected from polluted Ennore estuary showed increased levels of lipid peroxide and mtHSP70 expression along with decrease in total antioxidant capacity and glutathione redox ratio levels (^c P < 0.05) when compared to unpolluted Kovalam estuary fish. In the fish liver mitochondria of Ennore estuary, there was significant seasonal variation (^b P < 0.05) in both oxidative stress marker levels (34% increase) and mitochondrial HSP70 expression (33% increase) with increased level during summer season but the Kovalam estuary fish did not show any significant seasonal variation. In the Ennore estuary fish that are exposed to chronic environmental stress, the overexpression of liver mtHSP70 particularly during summer season may confer differential effects on the cell survival by protection against oxidative stress induced changes.     

Historical metal pollution in natural gudgeon populations: Inferences from allozyme, microsatellite and condition factor analysis

This study presents the results of a microsatellite and allozyme analysis on natural populations of the gudgeon (Gobio gobio) located in a pollution gradient of cadmium and zinc. Differences among contaminated and reference populations were observed at 2 allozyme loci, as well as a relationship between the fish condition factor and glucose-6-phosphate dehydrogenase genotypes, the locus that showed the largest difference in allele frequencies. The microsatellite data partly confirmed the differentiation pattern that was revealed by the allozyme survey. Our data further suggest that at least 2 microsatellite loci may be affected by natural selection. We thus illustrate that both microsatellite and allozyme loci do not necessarily behave as selectively neutral markers in polluted populations. Estimates of population differentiation can therefore be significantly different depending on which loci are being studied. Finally, these results are discussed in the light of the conservation unit concept, because microsatellites are often used to assess genetic variation in endangered natural populations and to propose measures for conservation or management.     

Heteroplasmic mitochondrial disease in Dictyostelium discoideum

The bewildering complexity of the relationship between genotype and phenotype in human mitochondrial diseases has delayed an understanding of the related cytopathological mechanisms. To explore the relationship between mitochondrial dysfunction in Dictyostelium discoideum and the related cytopathologies, we determined whether the phenotypic outcomes were similar regardless of which D. discoideum mitochondrial gene was targeted for disruption. The disruption of the mitochondrial genes resulted in a similar pattern of phenotypes to those caused by other mitochondrial defects. These include impairment of phototaxis, multicellular development and growth on plates and in liquid medium. As the reduced growth rates could have been due to defective phagocytic or macropinocytic nutrient uptake, these processes were tested but found to be unaffected. Since mitochondria have been associated with Legionella pathogenesis of human macrophages, it was also determined if mitochondrially diseased Dictyostelium strains were better or worse than healthy cells at supporting the growth of Legionella pneumophila. The results revealed that the mitochondrially diseased strains supported greater L. pneumophila growth than the wild type Dictyostelium strain (AX2). Quantitative Northern blotting showed a significant reduction in the level of expression of the entire mitochondrial genome, regardless of which mitochondrial gene was targeted for disruption, suggesting a generalized deficiency in mitochondrial gene expression and function. The phenotypic outcomes were the same as those shown previously to result from chronic hyperactivity of the energy-sensing protein kinase, AMPK, after knockdown of mitochondrial chaperonin 60.     

Alterations within the quinolone resistance-determining regions of GyrA and ParC of Neisseria gonorrhoeae isolated in the Far East and the United States

The genetic mutations within the quinolone resistance-determining regions (QRDRs) of gyrA and parC of 234 Neisseria gonorrhoeae strains isolated in the Far East and the United States, which exhibited either clinically significant ciprofloxacin resistance (CipR) or intermediate ciprofloxacin resistance (CipI) were characterized. A number of GyrA/ParC amino acid alteration patterns were identified, the most prevalent alteration pattern among CipR isolates being GyrA-91,95/ParC-Asp-86->Asn (91,95/Asp-86->Asn). Isolates containing 91,95/Asp-86->Asn belonged to a number of A/S classes, penicillin/tetracycline resistance phenotypes, and plasmid profiles. These results strongly suggest that the continuing emergence of ciprofloxacin-resistant gonococci is not due to the spread of a single or a few strains but to numerous factors such as spread of existing strains, importation of new strains and, possibly, de novo development of ciprofloxacin resistance in previously susceptible strains.     

Responses of Chub (Leuciscus cephalus) Populations to Chemical Stress,Assessed by Genetic Markers,DNA Damage and Cytochrome P4501A Induction

Indicators of effects at the population level (genetic variation using allozymes) and early indicators of pollution (EROD activity and DNA strand break formation) were analysed in chub (Leuciscus cephalus) living in weakly and heavily contaminated stations of the Rhône River watershed. The genetic erosion was mainly detected in a fish population living in a contaminated small river system, through modifications in allelic and genotypic frequencies for PGM-2 locus and could be linked to a genetic bottleneck and to the reduced gene flow from upstream unable to maintain or restore the genetic diversity. In a contaminated large river system, the genetic diversity for PGM-2 and other loci was maintained and was probably the consequence of a high gene flow from upstream, linked to a sustained drift of larvae and juveniles in the system. A convergent increase of the frequency of the 90 allele at PGM-2 was observed in two contaminated stations compared with the reference station, this trend being confirmed on a more extensive geographic scale over the Rhône River basin. A high level of EROD activity was detected in both contaminated sites but only the fish in the large river system showed a significant DNA damage level compared to the reference population. The low DNA damage level and high hepato-somatic ratio characterized the impacted population of the small river system and could be associated to a chronic high-level exposure of fish to pollutants which selected individuals exhibiting a high level of DNA damage repair. In the two contaminated systems, some genotypes at the PGM-2 and EST-2 loci showed a low level of DNA damage and/or a high EROD activity and may be considered as being tolerant to pollutants. A higher tolerance of the most heterozygous fish was also detected in the contaminated large system and confirmed that a high level of heterozygosity may be necessary for survival in such a system.     

Toxicity evaluation of water samples collected near a hospital waste landfill through bioassays of genotoxicity piscine micronucleus test and comet assay in fish Astyanax and ecotoxicity Vibrio fischeri and Daphnia magna

In this study, we analyzed samples of water from a river and a lake located near a hospital waste landfill with respect to physico-chemical parameters and conducted bioassays of ecotoxicity using Vibrio fischeri and Daphnia magna, which are species commonly used to evaluate the water toxicity. We also evaluated damage to the genetic material of fish (Astyanax sp. B) that were exposed (96 h) to water from these two sites that were located near the tank ditch, using the alkaline comet assay and the piscine micronucleus test. Parameters including aluminum, manganese, biochemical oxygen demand, sulfide, conductivity, phenol, total coliforms and Escherichia coli counts, were above acceptable levels that have been established in environmental legislation. However, the toxicity bioassays that we carried out in Vibrio fischeri and Daphnia magna and the piscine micronucleus test in fish showed no immediate risk due to acute effects. Based on the results of the comet assay, however, it was possible to detect damage to genetic material in fish that were acutely exposed in the laboratory to water samples from the river and lake that are located near the trench septic tank. Thus, our results suggest that tests beyond those usually employed to test water toxicity, such as the comet assay we used in the fish, are required to assess the toxicity of water with greater accuracy.