Alteration of hepato-lipidomic homeostasis in A/J mice fed an environmentally relevant PFAS mixture.

In the present study, we have investigated liver lipid homeostasis and corresponding changes in transcript and functional product levels in A/J mice exposed to environmental relevant concentration of per- and polyfluoroalkyl substances (PFAS) mixture. Mice were fed environmentally relevant concentrations of a PFAS mixture during a period of 10 weeks. The concentrations of the 8 individual PFAS in the mixture were chosen based on measured concentrations in earthworms at a Norwegian skiing area. Our data show high liver accumulation of ∑PFAS in exposed mice, which paralleled significant elevation in body weight and hepatosomatic index (HSI) of male mice. UPC2 -MS/MS analysis in both positive and negative mode, respectively, indicated significant differences between control and exposure groups in the liver of exposed mice. Principal component analysis (PCA) of the features revealed separation of control and exposure groups in both sexes. From the significantly differential 207 lipids, only 72 were identified and shown to belong to eight different lipid classes. PCA of fatty acids (FAs) profile showed a clear separation between control and PFAS exposure groups in both female and male mice, with differential abundant levels of 5 and 4 hydrolyzed FAs, respectively. Transcript and protein analysis of genes associated with lipid homeostasis (ppar-α and ß, lxr-α and ß, rxr, fasn and srebp) showed that PFAS exposure produced sex- and individual response related alterations. Glutathione reductase (Gr) activity showed exposure-related changes in both female and male mice, compared with controls. Overall, the present study has demonstrated changes in lipid metabolism after PFAS exposure, showing that PFAS accumulation in the liver resulted to hepatotoxic effects, potential interference with membrane lipid profile and homeostasis, and oxidative stress. Given the structural similarity with FAs, interaction between PFAS and nuclear receptors such as PPARs may have severe consequences for general health and physiology in exposed animals and humans.

Effects of an environmentally relevant PFAS mixture on dopamine and steroid hormone levels in exposed mice.

In the present study, we investigated the dopaminergic and steroid hormone systems of A/J mice fed environmentally relevant concentrations of a perfluoroalkyl substance (PFAS) mixture over a period of 10 weeks. The PFAS mixture was chosen based on measured PFAS concentrations in earthworms at a Norwegian skiing area (Trondheim) and consisted of eight different PFAS. Dietary exposure to PFAS led to lower total brain dopamine (DA) concentrations in male mice, as compared to control. On the transcript level, brain tyrosine hydroxylase (th) of PFAS exposed males was reduced, compared to the control group. No significant differences were observed on the transcript levels of enzymes responsible for DA metabolism, namely - monoamine oxidase (maoa and maob) and catechol-O methyltransferase (comt). We detected increased transcript level for DA receptor 2 (dr2) in PFAS exposed females, while expression of DA receptor 1 (dr1), DA transporter (dat) and vesicular monoamine transporter (vmat) were not affected by PFAS exposure. Regarding the steroid hormones, plasma and muscle testosterone (T), 11-ketotestosterone (11-KT) and 17ß-estradiol (E2) levels, as well as transcripts for estrogen receptors (esr1 and esr2), gonadotropin releasing hormone (gnrh) and aromatase (cyp19) were unaltered by the PFAS treatment. These results indicate that exposure to PFAS doses, comparable to previous observation in earthworms at a Norwegian skiing area, may alter the dopaminergic system of mice with overt consequences for health, general physiology, cognitive behavior, reproduction and metabolism.

Expression of DNA repair genes in arctic char (Salvelinus alpinus) from Bjørnøya in the Norwegian Arctic.

High levels of organochlorines (OCs) have been measured in arctic char (Salvelinus alpinus) from Lake Ellasjøen on Bjørnøya, Norway (74.30°N, 19.0°E). In a nearby lake, Laksvatn, the OC-levels in arctic char were low. A previous study has shown that char from Ellasjøen had significantly higher levels of DNA double strand breaks (DSBs) than char from Lake Laksvatn. Even though there is increasing evidence of the genotoxic effects of OCs, little is known about the effects of OCs on the DNA repair system. The aim of the present study was to determine if the two main DNA DSB repair mechanisms, homologous recombination (HR) and non-homologous end-joining (NHEJ), are affected by the higher OC and DSB level in char from Ellasjøen. This was analysed by comparing the transcript level of 11 genes involved in DNA DSB repair in char liver samples from Ellasjøen (n = 9) with char from Laksvatn (n = 12). Six of the investigated genes were significantly upregulated in char from Ellasjøen. As the expression of DNA DSB repair genes was increased in the contaminant-exposed char, it is likely that the DNA DSB repair capacity is induced in these individuals. This induction was positively correlated with the DNA DSB and negatively correlated with one or several OCs for four of these genes. However, the strongest predictor variable for DNA repair genes was habitat, indicating genetic differences in repair capacity between populations. As char from Ellasjøen still had significantly higher levels of DSBs compared to char from Laksvatn, it is possible that chronic exposure to OCs and continued production of DSB has caused selective pressure within the population for fixation of adaptive alleles. It is also possible that DSB production was exceeding the repair capacity given the prevailing conditions, or that the OC or DSB level was above the threshold value of inhibition of the DNA repair system resulting in the rate of DNA damage exceeding the rate of repair.

Alteration of neuro-dopamine and steroid hormone homeostasis in wild Bank voles in relation to tissue concentrations of PFAS at a Nordic skiing area.

Perfluoroalkyl substances (PFAS) are contaminants that are applied in a wide range of consumer products, including ski products. The present study investigated the neuro-dopamine (DA) and cellular steroid hormone homeostasis of wild Bank voles (Myodes glareolus) from a skiing area in Norway (Trondheim), in relation to tissue concentrations of PFAS. We found a positive association between brain DA concentrations and the concentration of several PFAS, while there was a negative association between PFAS and dopamine receptor 1 (dr1) mRNA. The ratio between DA and its metabolites (3,4-dihydroxyphenylacetic acid: DOPAC and homovanillic acid: HVA) showed a negative association between DOPAC/DA and several PFAS, suggesting that PFAS altered the metabolism of DA via monoamine oxidase (Mao). This assumption is supported by an observed negative association between mao mRNA and PFAS. Previous studies have shown that DA homeostasis can indirectly regulate cellular estrogen (E2) and testosterone (T) biosynthesis. We found no association between DA and steroid hormone levels, while there was a negative association between some PFAS and T concentrations, suggesting that PFAS might affect T through other mechanisms. The results from the current study indicate that PFAS may alter neuro-DA and steroid hormone homeostasis in Bank voles, with potential consequences on reproduction and general health.

Circulating trace elements: Comparison between early and late incubation in common eiders (Somateria mollissima) in the central Baltic Sea.

We analyzed body mass and a panel of 64 trace elements in blood from incubating common eiders (Somateria mollissima) in the central Baltic Sea during the breeding seasons of 2017 (n = 27) and 2018 (n = 23). Using a non-invasive approach, the same incubating eiders nesting on Christiansø, Denmark were sampled once on day 4 and day 24 of incubation to provide a comparison between the early and late stages of incubation. Blood concentrations of chemical elements were quantified using high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). Cadmium and lead significantly increased over the course of the incubation period while body mass, barium, calcium, cerium, cesium, iron, magnesium, manganese, molybdenum, phosphorus, selenium, strontium, sulfur, uranium, and zinc all significantly decreased. Excluding lead, all trace elements were within expected ranges. Lead blood concentrations had a 4.7-fold increase from 2017 to 2018 indicating a potential health threat. However, internal interactions between trace elements must be considered when making comparisons to toxicological thresholds. Body mass and many essential elements showed significantly higher levels in 2017 than 2018, which could be an indication of limitations in preferred food availability or harsher fasting conditions. Additional sampling years are needed to further investigate if these results reflect yearly fluctuations or decreasing health within the Christiansø eider colony. There was little overlap in element blood concentrations and body mass between days of incubation, indicating these parameters are affected by the physiological processes of reproduction and incubation. We recommend continued biomonitoring and use of complete trace element analysis for the Christiansø eiders to further understand year-to-year variations within colonies. Further investigation into the spatial ecology of the colony is also needed to provide a more robust understanding of exposure and source identification of trace elements.

Lead isotopic signatures in blood from incubating common eiders (Somateria mollissima) in the central Baltic Sea.

The Christiansø colony of common eiders (Somateria mollissima) in the central Baltic Sea were exposed to high levels of Pb during the 2018 breeding season that were not present in 2017. Due to these high Pb blood levels, the present study investigated possible Pb sources and Pb dynamics within this vulnerable colony. We analyzed body mass and lead isotopic ratios (Pb-IRs) in blood taken from the same incubating eiders at the early (day 4) and late (day 24) stages of incubation during the 2018 breeding season (n = 23). Pb-IRs 208/207, 208/206, 206/207, and 207/206 were analyzed using high resolution inductively coupled mass spectrometry. We found largely similar Pb-IRs from the different stages of incubation indicating a predominantly constant endogenous source of Pb exposure. We suggest the increasing Pb levels come from pre-nesting and nesting foraging and from medullary bone release. The similar Pb-IRs also indicate continued metabolization of the medullary bone to meet the nutritional and energy demands of incubation. Comparisons to Pb-IR reports from the Baltic Sea showed multiple sources of pollution distinguished by a difference between Pb-IRs in individuals with Pb blood concentrations >500 µg/kg ww and <500 µg/kg ww. The most highly contaminated individuals in the present study had Pb-IRs similar to those of Pb ammunition indicating shot pellet uptake. This study further emphasizes the need for continued biomonitoring of the Christiansø colony, including fecal sampling and environmental field sampling to identify the origin and extent of dietary Pb exposure on Christiansø. As a representative unit of the Baltic Flyway population; the Christiansø colony provides an important opportunity for continued investigation into Pb contamination, population dynamics, and declines.

Lead concentrations in blood from incubating common eiders (Somateria mollissima) in the Baltic Sea.

Here we investigate if lead may be a contributing factor to the observed population decline in a Baltic colony of incubating eiders (Somateria mollissima). Body mass and blood samples were obtained from 50 incubating female eiders at the Baltic breeding colony on Christiansø during spring 2017 (n = 27) and 2018 (n = 23). All the females were sampled twice during early (day 4) and late (day 24) incubation. The full blood was analysed for lead to investigate if the concentrations exceeded toxic thresholds or changed over the incubation period due to remobilisation from bones and liver tissue. Body mass, hatch date and number of chicks were also analysed with respect to lead concentrations. The body mass (mean ± SD g) increased significantly in the order: day 24 in 2018 (1561 ± 154 g) < day 24 in 2017 (1618 ± 156 g) < day 4 in 2018 (2183 ± 140 g) < day 4 in 2017 (2359 ± 167 g) (all p < 0.001). The lead concentrations increased significantly in the opposite order i.e. day 4 in 2017 (41.7 ± 67.1 µg/L) < day 24 in 2017 (55.4 ± 66.8 µg/L) < day 4 in 2018 (177 ± 196 µg/L) < day 24 in 2018 (258 ± 243) (all p < 0.001). From day 4 to 24, the eider females had a 1.33-fold increase in blood lead concentrations in 2017 and a 1.46-fold increase in 2018. Three of the birds (13%) sampled in 2018 had lead concentrations that exceeded concentrations of clinical poisoning (500 µg/L) and eleven (48%) had concentrations that exceeded the threshold for subclinical poisoning (200 µg/L). In 2017, none of the birds exceeded the high toxic threshold of clinical poisoning while only one (4%) exceeded the lower threshold for subclinical poisoning. Three of the birds (6%) sampled in 2018 had lead concentrations that exceeded those of clinical poisoning while 12 birds (24%) resampled in both years exceeded the threshold for subclinical poisoning. In addition, lead concentrations and body mass on day 4 affected hatch date positively in 2018 (both p < 0.03) but not in 2017. These results show that bioavailable lead in bone and liver tissue pose a threat to the health of about 25% of the incubating eiders sampled. This is particularly critical because eiders are largely capital breeding which means that incubating eiders are in an energetically stressed state. The origin of lead in incubating eiders in the Christiansø colony is unknown and it remains an urgent priority to establish the source, prevalence and mechanism for uptake. The increase in lead from day 4 to day 24 is due to bone and liver remobilization; however, the additional lead source(s) on the breeding grounds needs to be identified. Continued investigations should determine the origin, uptake mechanisms and degree of exposure to lead for individual birds. Such research should include necropsies, x-ray, lead isotope and stable C and N isotope analyses to find the lead sources(s) in the course of the annual cycle and how it may affect the population dynamics of the Christiansø colony which reflects the ecology of the Baltic eiders being suitable for biomonitoring the overall flyway.

Levels, Patterns, and Biomagnification Potential of Perfluoroalkyl Substances in a Terrestrial Food Chain in a Nordic Skiing Area.

Perfluoroalkyl substances (PFASs) are used in a wide range of consumer products, including ski products, such as ski waxes. However, there is limited knowledge on the release of PFASs from such products into the environment and the resultant uptake in biota and transport in food webs. We investigated levels, patterns, and biomagnification of PFASs in soil, earthworms (Eisenia fetida), and Bank voles (Myodes glareolus) from a skiing area in Trondheim, Norway. In general, there was higher PFAS levels in the skiing area compared to the reference area with no skiing activities. The skiing area was dominated by long-chained perfluorocarboxylic acids (PFCAs, ≥70%), while the reference area was dominated by short-chained PFCAs (>60%). The soil PFAS pattern in the skiing area was comparable to analyzed ski waxes, indicating that ski products are important sources of PFASs in the skiing area. A biomagnification factor (BMF) > 1 was detected for Bank volewhole/earthwormwhole for perfluorooctansulfonate in the skiing area. All other PFASs showed a BMF < 1. However, it should be noted that these organisms represent the base of the terrestrial food web, and PFASs originating from ski wax may result to higher exposure in organisms at the top of the food chain.

DNA Double-Strand Breaks in Arctic Char (Salvelinus alpinus) from Bjørnøya in the Norwegian Arctic.

High levels of organochlorine contaminants (OCs) have been found in arctic char (Salvelinus alpinus) from Lake Ellasjøen, Bjørnøya (Norwegian Arctic). The aim of the present study was to investigate the potential genotoxic effect of environmental organochlorine contaminant exposure in arctic char from Ellasjøen compared with arctic char from the low-contaminated Lake Laksvatn nearby. Blood was analyzed using agarose gel electrophoresis and image data analysis to quantify the fraction of total DNA that migrated into the gel (DNA-FTM) as a relative measure of DNA double-strand breaks (DSBs). Analysis by GC-MS of muscle samples showed an average 43 times higher concentration of ΣOCs in arctic char from Ellasjøen (n = 18) compared with Laksvatn char (n = 21). Char from Lake Ellasjøen had a much higher frequency of DSBs, as measured by DNA-FTM, than char from Lake Laksvatn. Principal component analysis and multiple linear regressions show that there was a significant positive relationship between DSBs and levels of organochlorine contaminants in the char. In addition, DSBs were less frequent in reproductively mature char than in immature char. The results suggest that organochlorine contaminants are genotoxic to arctic char. Environ Toxicol Chem 2019;38:2405-2413. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Biotransformation and oxidative stress responses in rat hepatic cell-line (H4IIE) exposed to racemic ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP).

Pharmaceuticals such as racemate ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP) are highly detectable non-steroidal anti-inflammatory drugs (NSAIDs) in the aquatic environment and therefore are designated as one of the most emerging groups of pollutants that can affect environmental and human health. The potential impact of these pharmaceuticals was assessed for the first time in vitro using a rat hepatocellular carcinoma cell line (H4IIE). Cells were exposed to low and high concentrations of these drugs. Cytotoxicity was determined by MTT reduction assay; CYP1A1 transcriptional and enzymatic levels together with canonical oxidative stress responsive markers (GPx, GR, GST and CAT) were also investigated. Cells exposed to RS-KP and S(+)-KP did not show cytotoxicity effect at the concentrations tested. However, this study highlighted differences between RS-KP and S(+)-KP in most of the evaluated markers, showing compound-, concentration- and time-specific effect patterns which suggest a potential stereo-selective toxicity of these drugs.

Blood and feather concentrations of toxic elements in a Baltic and an Arctic seabird population.

We report blood and feather concentrations of elements in the Baltic Sea and Arctic population of common eiders (Somateria mollissima). The endangered Baltic Sea population of eiders was demonstrably affected by element pollution in the 1990s. While blood concentrations of Hg were higher in Baltic breeding eiders, blood Se, As and Cd concentrations were higher in Arctic eiders. Blood concentrations of Pb, Cr, Zn and Cu did not differ between the two populations. While blood Pb concentrations had declined in Baltic eiders since the 1990s, Hg concentrations had not declined, and were above concentrations associated with adverse oxidative effects in other bird species. Inconsistent with blood concentrations, feather concentrations suggested that Pb, Zn, and Cd exposure was higher in Baltic eiders, and that Hg exposure was higher in Arctic eiders. Our study thus emphasizes the need for comprehensive evaluation of toxic element status, covering the annual cycle of a species.

Exposure of juvenile turbot (Scophthalmus maximus) to silver nanoparticles and 17α-ethinylestradiol mixtures: Implications for contaminant uptake and plasma steroid hormone levels.

Combined exposure to engineered nanoparticles (ENPs) and anthropogenic contaminants can lead to changes in bioavailability, uptake and thus effects of both groups of contaminants. In this study we investigated effects of single and combined exposures of silver (Ag) nanoparticles (AgNPs) and the synthetic hormone 17α-ethinylestradiol (EE2) on tissue uptake of both contaminants in juvenile turbot (Scophthalmus maximus). Silver uptake and tissue distribution (gills, liver, kidney, stomach, muscle and bile) were analyzed following a 14-day, 2-h daily pulsed exposure to AgNPs (2 µg L-1 and 200 µg L-1), Ag+ (50 µg L-1), EE2 (50 ng L-1) and AgNP + EE2 (2 or 200 µg L-1+50 ng L-1). Effects of the exposures on plasma vitellogenin (Vtg) levels, EE2 and steroid hormone concentrations were investigated. The AgNP and AgNP + EE2 exposures resulted in similar Ag concentrations in the tissues, indicating that combined exposure did not influence Ag uptake in tissues. The highest Ag concentrations were found in gills. For the Ag+ exposed fish, the highest Ag concentrations were measured in the liver. Our results show dissolution processes of AgNPs in seawater, indicating that the tissue concentrations of Ag may partly originate from ionic release. Plasma EE2 concentrations and Vtg induction were similar in fish exposed to the single contaminants and the mixed contaminants, indicating that the presence of AgNPs did not significantly alter EE2 uptake. Similarly, concentrations of most steroid hormones were not significantly altered due to exposures to the combined contaminants versus the single compound exposures. However, high concentrations of AgNPs in combination with EE2 caused a drop of estrone (E1) (female fish) and androstenedione (AN) (male and female fish) levels in plasma below quantification limits. Our results indicate that the interactive effects between AgNPs and EE2 are limited, with only high concentrations of AgNPs triggering synergistic effects on plasma steroid hormone concentrations in juvenile turbots.

DNA double-strand breaks in incubating female common eiders (Somateria mollissima): Comparison between a low and a high polluted area.

Alterations in the genetic material may have severe consequences for individuals and populations. Hence, genotoxic effects of environmental exposure to pollutants are of great concern. We assessed the impact of blood concentrations of persistent organic pollutants (POPs) and mercury (Hg) on DNA double-strand break (DSB) frequency, in blood cells of a high-exposed Baltic, and lower exposed Arctic population of common eiders (Somateria mollissima). Furthermore, we examined whether the genotoxic response was influenced by antioxidant concentration (plasma total glutathione (tGSH) and total antioxidant capacity) and female body mass. The DNA DSB frequency did not differ between the two populations. We found significant positive relationships between Hg and DNA DSB frequency in Baltic, but not in Arctic eiders. Although both p,p'-DDE and PCB 118 had a lesser effect than Hg, they exhibited a positive association with DNA DSB frequency in Baltic eiders. Antioxidant levels were not important for the genotoxic effect, suggesting alternative mechanisms other than GSH depletion for the relationship between Hg and DNA DSBs. Hence, the Baltic population, which is considered to be endangered and is under the influence of several environmental stressors, may be more susceptible to genotoxic effects of environmental exposure to Hg than the Arctic population.

Antioxidant Responses in Relation to Persistent Organic Pollutants and Metals in a Low- and a High-Exposure Population of Seabirds.

Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense. Exposure to pollutants may increase ROS and affect antioxidant levels, and the resulting oxidative stress may negatively affect both reproduction and survival. We measured concentrations of 18 persistent organic pollutants (POPs) and 9 toxic elements in blood, as well as total antioxidant capacity (TAC), total glutathione (tGSH), and carotenoids in plasma of Baltic and Arctic female common eiders (Somateria mollissima) (N = 54) at the end of their incubation-related fasting. The more polluted Baltic population had higher TAC and tGSH concentrations compared to the Arctic population. Carotenoid levels did not differ between populations. The effect of mixtures of pollutants on the antioxidants was assessed, and the summed molar blood concentrations of 14 POPs were positively related to TAC. There was no significant relationship between the analyzed pollutants and tGSH concentrations. The adaptive improvement of the antioxidant defense system in the Baltic population may be a consequence of increased oxidative stress. However, both increased oxidative stress and energy allocation toward antioxidant defense may have adverse consequences for Baltic eiders at the incubation stage, when energy resources reach an annual minimum due to incubation-related fasting.

Persistent organic pollutant levels and the importance of source proximity in Baltic and Svalbard breeding common eiders.

The distance to sources and the long-range transport potential of persistent organic pollutants (POPs) are important in understanding the impact of anthropogenic pollution on natural seabird populations. The present study documented blood concentrations of POPs in the Baltic Sea (Tvärminne, Finland) population of common eiders (Somateria mollissima) in 2009 and in 2011 and compared the concentrations with the presumably less exposed Arctic population in Svalbard (Kongsfjorden, Norway). The Baltic population had 26, 10, and 5 times greater concentrations of hexachlorocyclohexane, polychlorinated biphenyls, and p,p'-dichlorodiphenyldichloroethylene than the Svalbard population. Unexpectedly, concentrations of chlordanes were higher in Svalbard eiders, whereas concentrations of hexachlorobenzenes (HCBs) did not differ between the 2 populations. Although the similar HCB levels may partly be explained by the high transport potential of HCBs, unknown factors may have been more important than distance to sources and long-range transport potential for the chlordanes. One plausible explanation may be that the fasting-related redistribution of POPs from fat to blood was greater throughout the incubation in Arctic eiders, causing them to have higher blood levels of these POPs at the end of incubation. The blood concentrations of POPs in Baltic eiders were higher than documented in any other eider population and were comparable to levels in seabirds feeding at higher trophic positions in the food chain. Environ Toxicol Chem 2016;35:1526-1533. © 2015 SETAC.

DNA double-strand breaks in relation to persistent organic pollutants in a fasting seabird.

Lipophilic persistent organic pollutants (POPs) are released from fat reserves during fasting, causing increased blood concentrations. Thus, POPs represent a potential anthropogenic stressor during fasting periods. We analysed the blood of female common eiders (Somateria mollissima) by using agarose gel electrophoresis and image data analysis to quantify the DNA-fraction, of total DNA, that migrated into the gel (DNA-FTM) as a relative measure of DNA double strand-breaks (DSBs) during the fasting incubation period in the high arctic. In 2008 and in 2009 blood samples were obtained for analysis of 9 POPs and DNA-FTM at day 5 of the incubation period, and then in the same individuals at day 20. This unique study design gave us the opportunity to analyse the same individuals throughout two points in time, with low and high stress burdens. During the incubation period the body mass (BM) decreased by 21-24%, whereas the POP levels increased by 148-639%. The DNA-FTM increased by 61-67% (being proportional to the increase in DSBs). At day 5, but not day 20, DNA-FTM was positively correlated with most analysed POPs. The increase in DNA-FTM was positively correlated with the decrease in BM (g) during incubation. Thus, we suggest that fasting stress (BM loss) decreases DNA integrity and that stress caused by fasting on BM loss appeared to override the additional stress caused by concurrent increase in levels of the analysed POPs in the eiders. Blood levels of POPs in the eiders in Svalbard were relatively low, and additive and/or synergistic genotoxic effects of fasting stress and POP exposure may occur in populations with higher POP levels.

Induction of DNA double-strand breaks in the H4IIE cell line exposed to environmentally relevant concentrations of copper, cadmium, and zinc, singly and in combinations.

Xenobiotics, including heavy metals, exist in nature as complex mixtures of compounds with possible interactions. Induction of DNA damage such as DNA strand breaks may exert detrimental consequences to both individuals and populations. In this study, the induction of DNA double-strand breaks was assessed using the H4IIE rat hepatoma cell line following exposure to high and environmentally relevant concentrations of chloride salts of the metals cadmium (Cd), copper (Cu), and zinc (Zn), both singly and in combination. DNA strand break analysis was performed using agarose gel electrophoresis. Median molecular lengths were calculated from fragment size distributions acquired from gel image data and were used as a quantitative measure of DNA double-strand break induction. Exposure to high concentrations of Cu and Cd in combination produced a significant increase in the occurrence of DNA strand break. However, exposing cells to high concentrations of Cu, Cd, and Zn in combination resulted in significantly lower DNA double-strand break compared to control cells. Addition of low Zn to the Cd/Cu mixture restored DNA damage level back to that of the control. Environmentally relevant concentrations of Cd, Cu, and Zn did not appear to induce DNA strand breaks in the H4IIE cell line.

Application of statistical experimental design and multivariate data analysis for evaluation of mixtures using cytochrome P4501A induction.

Cytochrome P4501A (CYP1A) induction was evaluated in the rat hepatoma cell line H4IIE as a biomarker of exposure to organic compounds. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay was performed to assess the viability of cells exposed to environmentally relevant concentrations of cadmium. Cadmium concentrations greater than approximately 0.7 microM were found to affect cell viability. Cells were exposed to environmentally relevant concentrations of 3,3',4,4'-tetrachlorobiphenyl (PCB 77) or benzo[a]pyrene (BaP) and to combinations of PCB 77, BaP, and cadmium based on a statistical experimental design. Quantification of CYP1A proteins using Western blot analysis showed that both BaP and PCB 77 induced CYP1A in a concentration-dependent manner up to 5 microM. Response surface modeling for evaluation of the combined effect of compounds was conducted using the multivariate regression model projection to latent structures (PLS). Analysis of response surface models for the ternary mixtures indicated antagonistic interactions between BaP and PCB 77 and a possible inhibitory effect of cadmium on PCB 77- induced CYP1A. Use of CYP1A induction in the H4IIE cell line with immunodetection of CYP1A proteins, combined with the application of response surface design and PLS, is shown to be a suitable strategy for evaluating combined effects in pollutant mixtures.

Chromosome aberrations and DNA strand breaks in glaucous gull (Larus hyperboreus) chicks fed environmentally contaminated gull eggs.

In this present laboratory study, our results suggest that a complex mixture of pollutants found in the marine environment exerts genotoxic effects on glaucous gull (Larus hyperboreus) chicks fed environmentally contaminated gull eggs. Chromosome aberrations, quantified by cytogenetic analysis of blood cells, and DNA strand breaks, quantified by agarose gel electrophoresis and image data analysis, were determined in glaucous gull chicks fed environmentally contaminated gull eggs (exposed group) and in chicks fed hen eggs (control group). For both female and male gulls, the fraction of damaged metaphases was quantitatively higher in exposed than in control groups. On the other hand, the differences between the control and the exposed groups were more relevant when the chromosomal aberration data were treated as group totals rather than at the individual level. Consistent results were obtained in the DNA strand break analyses. The control group appeared to display a greater median molecular length (MML) than the exposed group.

Cytochrome P4501A induction and DNA adduct formation in glaucous gulls (Larus hyperboreus), fed with environmentally contaminated gull eggs.

This study indicates that complex mixtures of pollutants found in the Arctic marine environment have genotoxic effects in glaucous gulls (Larus hyperboreus). DNA adducts were quantified, by the (32)P-postlabeling technique, in liver samples from gulls fed with hen eggs (controls) and from gulls fed with environmentally contaminated gull eggs (exposed). All birds were grown and fed under laboratory conditions. Hepatic homologues to mammalian cytochrome P4501A (CYP1A) proteins were also determined by Western blotting. DNA adducts were detected in all but one liver sample, but the exposed birds had a significantly increased level of DNA adducts relative to that of the controls. There was no clear significant correlation between the DNA adduct level and the level of organochlorine compounds (OCs) in blood. The level of CYP1A protein was significantly higher in the liver of exposed male gulls than in the liver of control males and positively correlated, with significance, to the level of OC compounds measured in blood. There was no significant correlation between the level of DNA adducts and the CYP1A protein content.