Radiation-induced adaptive response in fish cell lines

There is considerable interest at present in low-dose radiation effects in non-human species. In this study gamma radiation-induced adaptive response, a low-dose radiation effect, was examined in three fish cell lines, (CHSE-214 (Chinook salmon), RTG-2 (rainbow trout) and ZEB-2J (zebrafish)). Cell survival after exposure to direct radiation with or without a 0.1 Gy priming dose, was determined using the colony forming assay for each cell line. Additionally, the occurrence of a bystander effect was examined by measuring the effect of irradiated cell culture medium from the fish cell lines on unexposed reporter cells. A non-linear dose response was observed for all cell lines. ZEB-2J cells were very sensitive to low doses and a hyper-radiosensitive (HRS) response was observed for doses <0.5 Gy. A typical protective adaptive response was not detected in any of the three fish cell lines tested. Rather, it was found that pre-exposure of these cells to 0.1 Gy radiation sensitized the cells to subsequent high doses. In CHSE-214 cells, increased sensitivity to subsequent high doses of radiation was observed when the priming and challenge doses were separated by 4 h; however, this sensitizing effect was no longer present when the interval between doses was greater than 8 h. Additionally, a "protective" bystander response was observed in these cell lines; exposure to irradiated medium from fish cells caused increased cloning efficiency in unirradiated reporter cells. The data confirm previous conclusions for mammalian cells that the adaptive response and bystander effect are inversely correlated and contrary to expectations probably have different underlying mechanisms.     

Comparison of transfer and effects of Cd on rats exposed in a short experimental snail-rat food chain or to CdCl2 dosed food

Transfer and toxic effects of two cadmium (Cd) forms, inorganic (CdCl2 dosed rat food) or organic (contaminated snail-based rat food) were studied in Wistar rat. Cd concentrations in rat food were 0 and 2.5 microg Cd g(-1) for both inorganic and organic forms and a high concentration of 100 microg Cd g(-1) was also tested for the inorganic form. Rats were exposed for four weeks to contaminated food. Both forms of Cd were bioavailable to rats, with a percentage of transfer from food to rats of around 1% for all contaminated groups. Cd concentrations in rat tissues increased with increasing Cd concentrations in the food. Rats fed with organic form of Cd accumulated significantly more Cd in the main organ for Cd toxicity, the kidney, than those eating the inorganic form. Survival was not affected for any rat group but a decrease in growth and food consumption was observed for the inorganic form. As a defence system against Cd toxicity, rats increased their metallothionein (MT) synthesis at the highest Cd concentration in the target organs (kidney, liver and small intestine) and even did the same at low Cd concentrations (2.5 microg Cd g(-1)) in the kidney. At this low Cd concentration, MT induction was lower in the small intestine of rats ingesting organic Cd than those ingesting inorganic Cd. Bioavailability of organic and inorganic forms of Cd was similar, but subsequent Cd distribution within organs was different. This quantification of the trophic transfer of both inorganic and organic forms of a toxicant is a basis for a better assessment of the fate and effects of chemicals in food webs.     

Comparative accumulation of (109)Cd and (75)Se from water and food by an estuarine fish (Tetractenos glaber)

Few data are available on the comparative accumulation of metal(loid)s from water and food in estuarine/marine fish. Smooth toadfish (Tetractenos glaber), commonly found in estuaries in south-eastern Australia, were separately exposed to radio-labelled seawater (14kBqL(-1) of (109)Cd and 24kBqL(-1) of (75)Se) and food (ghost shrimps; Trypaea australiensis: 875Bqg(-1)(109)Cd and 1130Bqg(-1)(75)Se) for 25 days (uptake phase), followed by exposure to radionuclide-free water or food for 30 days (loss phase). Toadfish accumulated (109)Cd predominantly from water (85%) and (75)Se predominantly from food (62%), although the latter was lower than expected. For both the water and food exposures, (109)Cd was predominantly located in the gut lining (60-75%) at the end of the uptake phase, suggesting that the gut may be the primary pathway of (109)Cd uptake. This may be attributed to toadfish drinking large volumes of water to maintain osmoregulation. By the end of the loss phase, (109)Cd had predominantly shifted to the excretory organs - the liver (81%) in toadfish exposed to radio-labelled food, and in the liver, gills and kidney (82%) of toadfish exposed to radio-labelled water. In contrast, (75)Se was predominantly located in the excretory organs (gills, kidneys and liver; 66-76%) at the end of the uptake phase, irrespective of the exposure pathway, with minimal change in percentage distribution (76-83%) after the loss phase. This study emphasises the importance of differentiating accumulation pathways to better understand metal(loid) transfer dynamics and subsequent toxicity, in aquatic biota.     

Complex toxic effects of Cd2+, Zn2+, and acid rain on growth of kidney bean (Phaseolus vulgaris L)

Complex toxic effects of Cd2+, Zn2+, and acid rain on growth of kidney bean (Phaseolus vulgaris L) were studied in a pot experiment by measurement of fresh weights of the plants, determination of surperoxide dismutase (SOD), peroxidase (POD), and lipid peroxidation (MDA) in the plant organs, and observation of injury symptoms. The experimental results demonstrated that all treatments of Cd2+, Zn2+, and/or acid rain significantly decreased fresh weights of kidney bean and caused toxic effects on growth of the plants, especially higher amounts of Cd2+ and Zn2+ and higher acidity of acid rain. Combination of these three pollutant factors resulted in more serious toxic effects than any single pollutant and than combinations of any two pollutants. SOD, POD, and MDA in the plant organs changed with different pollution levels, but MDA content in the leaves showed the best relationship between the pollution levels and toxic effects.     

Interspecific distribution and co-associations of chemical elements in the liver tissue of marine mammals from the Polish Economical Exclusive Zone, Baltic Sea

Concentrations of Al, B, Ba, Cd, Co, Cr, Cu, Fe, Ga, Hg, Li, Mn, Mo, Ni, Pb, Se, Si, Sr, Tl, V, Zn, Ca, K, Mg, Na and P in the livers of marine mammals obtained from by-catches or stranded on beaches on the Polish Baltic coast were determined by ICP-MS or ICP-AES and CV AAS. Interspecific diversity with respect to the contents of these elements was found in cetaceans and pinnipeds. The diverse Cd contents in the livers of these mammals can be attributed to the variable concentrations of this element in their food. Mercury was correlated with age and can reach high concentration associated with higher level of Se in older specimens. No significant relationships were found between concentration of the chemical elements studied and nutritional status/condition of the Baltic harbour porpoises as well as between their concentration in specimens from the Gulf of Gdańsk and open Baltic. It seems that the nutritional and health status of the specimens studied is generally enough good since the specimens studied were not stranded on beach because of starvation but almost incidentally caught in salmon gill nets. Strong correlations were found between the macroelements analysed, i.e. for the Ba-Ca-Sr, Ca-K, Ca-Mg, Mg-P, Zn-Mg and Zn-P assemblies. Significantly higher content of Al was found in males of harbour porpoises.     

Metal and metallothionein content in tissues from wild and farmed Anguilla anguilla at commercial size

Metallothionein and metal content (Cd, Zn, Hg, Cu, Fe, Pb and Mn) were determined in various organs of commercially available eel (Anguilla anguilla) of similar size obtained from a local farm and from The Albufera Lake in Valencia (Spain). Farmed fish showed statistically significant higher Cd concentrations in liver and kidney whereas wild individuals had higher levels of Pb in blood and Zn in kidney. Significant positive correlations were found between metallothionein and Cd in kidney of farmed eel and between metallothionein and Cu in liver of wild ones. No statistically significant differences were found between the two populations in the concentration of any of the metals analyzed in muscle and in all instances these levels were lower than the limits established by the Spanish legislation for fish destined for human consumption.     

Fly ash leachate induces oxidative stress in freshwater fish Channa punctata (Bloch)

Oxidative stress inducing potential of fly ash leachate (FAL) was studied in a freshwater fish, Channa punctata (Bloch). Fish were exposed to fly ash leachate for 24 h and lipid peroxidation (LPO) was studied as a marker of oxidative stress. Catalase (CAT), glutathione S-transferase (GST) activities and levels of reduced glutathione (GSH) were also estimated in the exposed fish. FAL (1 ml/l) induced LPO in all the organs and most prominent response was in the gill. It also caused induction of enzymes and glutathione. Liver showed highest level of induction of enzyme activities. The results of this study demonstrate that fly ash constituents have potential to induce oxidative stress in fish and gills are the most vulnerable organs. It is also suggested that in case of exposure to FAL, along with LPO antioxidant defense is also activated to counteract the reactive oxygen species (ROS) at least partly in the initial stages of exposure.     

蒌蒿(Artemisia selengensis L.)修复洞庭湖土壤Cd污染的强化措施研究

〖HTSS〗洞庭湖土壤镉污染严重,蒌蒿（Artemisia selengensis L.）是在洞庭湖湿地新发现的一种对镉具有较强富集能力的优势植物,已证实该种植物对土壤中的镉具有较好的修复效果和潜能,将来可作为洞庭湖土壤镉污染的理想修复材料。以南洞庭湖Cd污染土壤为栽培基质,分别在蒌蒿幼苗期、成株期添加EDTA等7种螯合剂和调控物质,研究了不同添加物对蒌蒿生长状况及Cd富集效果的影响。结果表明,HEDTA、EDTA、DTPA等3种螯合剂不同程度地降低了蒌蒿地上部分的生物量,但增加了土壤中有效态Cd的含量,使蒌蒿茎叶中Cd的富集浓度分别上升了35.5%、984%、421%,可显著提高蒌蒿的修复效果;生石灰的添加则使土壤有效态Cd明显减少,抑制了蒌蒿对Cd的富集;有机腐殖质和复合肥的施加虽促进了蒌蒿生长,但未对其富集效果产生显著影响;幼苗期施加调控物质,在植株生物量、Cd富集浓度及土壤有效态Cd含量等方面造成的影响均大于成株期     

The use of the oxidative stress responses as biomarkers in Nile tilapia (Oreochromis niloticus) exposed to in vivo cadmium contamination

Water contaminants have a high potential risk for the health of populations. Protection from toxic effects of environmental water pollutants primarily involves considering the mechanism of low level toxicity and likely biological effects in organisms who live in these polluted waters. The biomarkers assessment of oxidative stress and metabolic alterations to cadmium exposure were evaluated in Nile tilapia, Oreochromis niloticus. The fish were exposed to 0.35, 0.75, 1.5, and 3.0 mg/l concentrations of Cd2+ (CdCl2) in water for 60 days. Fish that survived cadmium exposure showed a metabolic shift and a compensatory development for maintenance of the body weight gain. We observed a decreased glycogen content and decreased glucose uptake in white muscle. Lactate dehydrogenase (LDH) and creatine phosphokinase (CK) activities were also decreased, indicating that the glycolytic capacity was decreased in this tissue. No alterations were observed in total protein content in white muscle due to cadmium exposure suggesting a metabolic shift of carbohydrate metabolism to maintenance of the muscle protein reserve. There was an increase in glucose uptake, CK increased activity, and a clear increase of LDH activity in red muscle of fish with cadmium exposure. Since no alterations were observed in lipoperoxide concentration, while antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were changed in the liver and the red and white muscle of fish with cadmium exposure, we can conclude that oxygen free radicals are produced as a mediator of cadmium toxicity. Resistance development is related with increased activities of antioxidant enzymes, which were important in the protection against cadmium damage, inhibiting lipoperoxide formation.     

Biotransformation, stress and genotoxic effects of 17beta-estradiol in juvenile sea bass (Dicentrarchus labrax L.)

The effects of 17beta-estradiol (E(2)) on fish became a matter of concern, since significant levels of this hormone were detected in the aquatic environment released mainly by domestic sewage treatment plants. In this perspective, the current study was focused on E(2) effects upon biotransformation, stress and genotoxic responses of juvenile Dicentrarchus labrax L. (sea bass). Fish were exposed to E(2) during 10 days in two different ways: water diluted (200 ng/L or 2,000 ng/L) and i.p. injected (0.5 mg/kg or 5 mg/kg). A battery of biological responses was evaluated: liver ethoxyresorufin-O-deethylase (EROD) and alanine transaminase (ALT) activities, liver somatic index (LSI), plasma cortisol, glucose and lactate concentrations, as well as erythrocytic nuclear abnormalities (ENA). All the exposure conditions induced endocrine disruption, measured as plasma cortisol decrease, and genotoxicity, measured as ENA increase. Thus, no differences were detected either between different exposure routes or tested concentrations. Concerning liver EROD and ALT activities, as well as plasma glucose and lactate concentrations no differences were found between treated and control groups. LSI was the only parameter to respond differently in the two exposure routes, as only E(2) water diluted induced a significant increase in this hepatic indicator.     

An in vitro/in vivo screening assay as a sensitive tool to assess endocrine disruptive activity in surface water

Adult male fathead minnow were exposed for 14 or 28-days under flow-through conditions to undiluted filtered water samples from the rivers Meuse and Rhine in the Netherlands. The experiment included two vessels per treatment each containing 10 fish and samples of five fish were taken after 14 and 28 days. Additional groups were exposed to 17alpha-ethinylestradiol (EE2) as a reference and untreated drinking water as a negative control. Major endpoints examined included induction of vitellogenin (VTG) synthesis, VTG mRNA activity, hepato- and gonadosomatic indices (HSI and GSI) and gonadal histology. No significant difference was recorded in body weight or mean GSI values between the various treatments. Only exposure to Meuse water resulted in significantly higher HSI means after 14 days. Histological examination showed no apparent effects on gonadal tissue except for eosinophilic blood plasma in fish exposed to Meuse water or EE2. After 14 and 28 days, elevated VTG and VTG mRNA levels were measured in most livers of the fish exposed to Meuse water, but not in the fish exposed to Rhine water. This was confirmed by measuring estrogenic responses in the in vitro ER CALUX assay. Induction of VTG synthesis proved to be the most sensitive endpoint in the Non Spawning Male Fish Assay for in vivo detection of bio-available estrogenic activity supplementary to a sensitive in vitro assay. The other endpoints examined varied too much and required a higher number of fish or replicates to achieve sufficient power for statistical testing making them less animal friendly.     

Heavy Metals in the Environment of the Middle Urals and Their Effects on the Organism

The kinetics of accumulation of essential and toxic trace elements (TE) in vitally important organs were studied in small mammals typical of the Middle and Southern Ural fauna. The results revealed a discrepancy between the increased uptake of Cd, Zn, Pb, and Cu by animals and the virtually constant concentrations of heavy metals (HMs) in their organs, which could be explained by the existence of special elimination systems activated upon excessive HM uptake. It is shown that HM accumulation in animal tissues is energy-dependent and species-specific, and the effects of highly toxic TE correlate with physiological processes.     

Effects of cadmium and zinc on larval growth and survival in the ground beetle,Pterostichus oblongopunctatus

Carabid beetles, like Pterostichus oblongopunctatus, living in metal contaminated areas may be exposed to elevated levels of metals within their diets. However, when compared to other second order consumers, they have one of the lowest observed levels of metals, indicating methods of detoxification to deal with such toxicants. In this study, we investigated if chronic, multigenerational exposure to metals leads to resistance to toxic metal concentrations, and if so, what are the costs associated with them. Adult organisms were collected from two sites, a polluted and a reference site near Olkusz, in southern Poland. These adults were immediately mated, and eggs were collected twice weekly to assess the effects in the larvae of the F(1) generation. Larvae were randomly exposed to one of four artificial mediums: control, 50 mg kg(-1) Cd, 500 mg kg(-1) Zn, and a combined treatment of 50 mg kg(-1) Cd and 500 mg kg(-1) Zn to investigate possible interactions. Individuals were sacrificed at 10, 30, and 40 days. Although metals were not accumulated in larvae (p>0.05), larvae fed the Cd or the Zn treatment grew significantly slower, and had the lowest survival rate (p<0.05) in respect to control. Out of metal treated animals, those on the combined treatment of CdZn grew the quickest and had the highest observed survival (p<0.05). Although previous studies have demonstrated changes in adult population parameters under chronic, multigeneration exposure to toxic metal concentrations, our study did not reveal any changes in the larval stage.     

Heavy Metals in Organs and Tissues of the European Black Vulture (Aegypius monachus): Dependence on Living Conditions

The distribution of Fe, Zn, Cu, Cd, Mn, Pb, and Ni in the organs and tissues (kidneys, liver, lungs, brain, heart, muscles, and feathers) of the European black vulture (Aegypius monachus) was studied. It was shown that heavy metals were not responsible for the mass mortality of these birds in southern Primorye in the winter of 2001–2002. The level of pollution with heavy metals of the range of European black vultures wintering in this region (northern China and western Mongolia) may be regarded as insignificant.     

Selenium and 17 other largely essential and toxic metals in muscle and organ meats of Red Deer (Cervus elaphus)--consequences to human health

Concentrations, composition and interrelationships of selenium and metallic elements (Ag, Ba, Cd, Co, Cr, Cs, Cu, Ga, Mn, Mo, Pb, Rb, Sb, Sr, Tl, V and Zn) have been examined in muscle and organ meats of Red Deer hunted in Poland. The analytical data obtained were also discussed in terms of Se supplementation and deficit to Deer as well as the benefits and risk to humans associated with the essential and toxic metals intake resulting from consumption of Deer meat and products. These elements were determined in 20 adult animals of both sexes that were obtained in the 2000/2001 hunting season from Warmia and Mazury in the north-eastern part of Poland. The whole kidneys contained Ba, Cd, Cr, Ga, Pb, Se, Sr and Tl at statistically greater concentrations than liver or muscle tissue from the same animal. Liver showed statistically greater concentrations of Ag, Co, Cu, Mn and Mo than kidneys or muscle tissue, and muscle tissue was richer in Zn, when compared to the kidneys or liver. Cs and Rb were similarly distributed between all three tissue types, while V was less abundant in liver than kidneys or muscle tissue. There were significant associations between some metallic elements retained in Red Deer demonstrated by Principal Component Analysis (PCA) of the data set. In organ and muscle meats (kidneys, liver and muscle tissue considered together) the first principal component (PC1) was strongly influenced by positively correlated variables describing Se, Ba and Cd and negatively correlated variables describing Ag, Co, Cs, Mn, Pb, Tl and V; PC2, respectively, by Cu, Mn and Mo (+) and Zn (-); PC3 by Ga (+) and PC4 by Sb (+). Selenium occurred in muscle tissue, liver and kidneys at median concentrations of 0.13, 0.19 and 4.0mg/g dry weight, respectively. These values can be defined as marginally deficient (< 0.6mg Se/kg liver dw) or satisfactory (≤ 3.0mg Se/kg kidneys dw) for the amount required to maintain the Deer's body condition and health, depending on the criterion for supplementation used. In terms of human nutritional needs, a relatively high selenium content of kidneys can be beneficial. The muscle meat, liver and kidneys of Red Deer can be considered as a very good source of essential Co, Cr, Cu, Mo, Mn, Se and Zn in the human diet. Lead is generally considered as toxic, and the concentrations found in Red Deer (via the food chain intake) were well below the European Union tolerance limit. Pb from the lead bullets can always create food hygienic problem, if not well recognized during sanitary inspection, and this was noted for one muscle meat sample in this study (5% surveyed). There is no tolerance limit of Cd in game animal meats. The median values of Cd noted in fresh muscle tissue, liver and whole kidneys were 0.07, 0.18, and 3.3mg/kg wet weight, respectively. Cd exists as a chemical element present at trace levels in plants and mushrooms in Deer's food chain in background (uncontaminated) areas. When these are consumed by the Deer, the amount of Cd sequestered with metallothioneins and retained in the organ and muscle meat in this study is low enough to be considered safe for human consumption.     

Juvenile sea bass (Dicentrarchus labrax L.) DNA strand breaks and lipid peroxidation response following 17beta-estradiol two mode of exposures

Juvenile Dicentrarchus labrax L. (sea bass) were exposed to water diluted 17beta-estradiol (E2) (200 ng/L and 2000 ng/L) and treated with intraperitoneal (i.p) injection E2 (0.5 mg/kg and 5 mg/kg) during 10 days in order to study its genotoxicity and peroxidative damage, measured as gill, blood, liver and kidney DNA integrity decrease using DNA strand breaks assay as well as liver, gill and kidney lipid peroxidation (LPO) respectively. Juvenile sea bass gill DNA integrity was significantly decreased for all E2 exposure conditions. However, no differences were detected either between different exposure routes or tested concentrations. Blood DNA integrity was significantly decreased by E2 5 mg/kg as well as by both water diluted E2 exposure conditions. The highest E2 dose (5 mg/kg) also promoted liver DNA integrity decrease. Liver and gill LPO significantly increased at the highest E2 i.p treatment. An increasing trend of gill and liver LPO, though statistically insignificant, was observed in D. labrax exposed to water diluted E2 in both tested concentrations. The current results demonstrated that DNA damage in juvenile sea bass is affected by the E2 exposure conditions, such as water diluted E2 versus i.p E2 injection since i.p E2 injection promoted higher genotoxicity effect, in terms of affected organs than water diluted E2. Moreover, the organ sensitivity to E2 was different, since gill showed more susceptibility than blood, liver and kidney. Concerning kidney LPO and DNA integrity no differences were found between treated and controls juvenile sea bass groups.     

Deleterious effects in mice of fish-associated methylmercury contained in a diet mimicking the Western populations' average fish consumption

Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. Only a few contradictory epidemiological studies are currently available examining the impact of fish consumption on human populations. In the present study, we wanted to address whether a diet mimicking the fish consumption of Western populations could result in observable adverse effects in mice, and whether beneficial nutriments from fish were able to counterbalance the deleterious effects of MeHg, if any. In Europe and the United States, fish consumption varies widely between countries, from 11 to 100 g fish/day. A mid-range value of 25 g fish/day corresponds to a fish contribution to the total diet of 1.25% on a dry weight basis. We decided to supplement a vegetarian-based mouse diet with 1.25% of lyophilized salmon flesh (SAL diet), or 1.25% of a blend of lyophilized cod, tuna, and swordfish (CTS diet). Total mercury contents were 1.15±0.15, 2.3±0.1 and 35.75±0.15 ng Hg/g of food pellets for the control, SAL and CTS diets, respectively. After two months feeding, the CTS diet resulted in significant observable effects as compared to the control and SAL diets, encompassing decreased body growth, altered behavioral performance and increased anxiety level, modification of mitochondrial respiratory protein subunit concentrations in kidney and brain structures, modified gene expression patterns in kidneys, liver and muscles, and a decrease of dopamine concentrations in the hypothalamus and striatum. Our findings have health implications, firstly because 1.25% of CTS flesh in the diet corresponds to an average exposure to MeHg below the WHO provisory tolerable weekly intake (PTWI) (1.6 μg MeHg/kg of body weight/week), and secondly because many people in Western populations, among them women of child-bearing age, are exceeding the PTWI value (for instance, 35% of the French population inhabiting the Atlantic and Mediterranean coasts).     

Trace element levels in foetus-mother pairs of short-beaked common dolphins (Delphinus delphis) stranded along the French coasts

Tissues of foetus-mother pairs of common dolphins (Delphinus delphis) stranded along the French coasts (Bay of Biscay and English Channel) were analysed for their Cd, Cu, Hg, Se and Zn contents. In the kidneys, foetal Cd levels were extremely low, and strong relationships between Cu and Zn suggested the involvement of metallothioneins since early foetal life. The results also indicated a limited maternal transfer of Hg during pregnancy since levels in the tissues of foetuses were below 1 microg g(-1) w.wt. However, hepatic Hg levels in foetuses increased with body length, and were also proportionate to maternal hepatic, renal and muscular Hg levels. Lastly, affinities between Hg and Se in tissues would participate in Hg neutralisation in both mothers--through tiemannite granules--and fetuses--through reduced glutathione--counteracting the toxic effects linked to the particularly high quantities of methyl-Hg to which marine mammals are naturally exposed.     

Effects of moderate pollution on toxic and trace metal levels in calves from a polluted area of northern Spain

This study evaluated the contribution of anthropogenic pollution to toxic metal residues (Cd, Pb and As) in cattle in an industrialized area of Asturias (northern Spain), and investigated possible implications of toxic metal exposure for metabolism of essential metal elements (Cu, Zn, Fe, Mn). Samples of liver, kidney, muscle and blood from animals aged 9-12 months were obtained from an industrialized area (78 calves) and from a rural area (92 calves). Samples were acid-digested, and levels of metals determined by atomic absorption spectrophotometry. Cadmium and lead contents in the liver and kidney were moderately and significantly higher in calves from the industrialized area (Cd: liver 29.6, kidney 161; Pb: liver 38.1, kidney 38.3 microg/kg wet weight) than in calves from the rural area (Cd: liver 22.9, kidney 96.4; Pb: liver 20.7, kidney 15.9 microg/kg kg wet weight). Although these toxic metals were only moderately raised, our results suggest that they may nevertheless have interfered with trace element metabolism, as reported previously for severely polluted regions. Notably, copper levels in calves from the industrialized area were generally low, and nearly half of these animals showed tissue copper levels indicating risk of copper deficiency.