A bioassay for metals utilizing a human cell line.

The purpose of this study was to assess the ability of the HepG2 cell line to function as a bioassay for metal contamination in sediments, using metallothionein (MT) as a biomarker of exposure. Sediments were collected from the eastern and western ends of Lake Erie, extracted using EPA method 200.7, and analyzed for cadmium (Cd), mercury (Hg) and lead (Pb) levels using ICP-AES. Sediment extracts were neutralized then used at a 2.5% final concentration in the exposure medium. MT levels were measured using the cadmium-hemoglobin affinity assay after a 48 h exposure. Fortified blanks from the ICP protocol served as positive controls. Also, HepG2 cells were exposed to Cd, Pb or combinations of Cd and Pb to determine whether or not induction of MT observed in cells exposed to sediment extracts was due to a single metal, combinations of metals, pH, or some other factor. Additionally, cells were exposed to a range of Cd concentrations approximating the levels found in the extracts (0.0005-0.1mg/L) to determine if a concentration-response occurred. Total metal levels ranged from 527 to 33.5mg/kg with lead the predominant metal, accounting for 100-88.9% of the total quantifiable metals in the sediments. The biomarker response (MT induction) was strongly correlated (r2=0.9919, r2=0.990) with total metal and lead levels in the sediments, respectively, which supports recent field studies indicating the biomarker can discern differences in the strength of the inducing agent. Statistically significant MT induction was associated with sediments which contained measurable Cd concentrations and no significant differences were observed when comparing Cd only and Cd+Pb exposed cells indicating no interactions between Cd and Pb were occurring and supporting our finding that Cd was the main inducing agent in sediment extracts. MT levels also increased significantly in a concentration-dependent manner when cells were exposed only to Cd. Results suggest this human bioassay and the MT biomarker of exposure may be useful for monitoring complex metal mixtures in aquatic sediments.

Organochlorine insecticides: impacts on human HepG2 cytochrome P4501A, 2B activities and glutathione levels.

This study examined the effects of the organochlorine (OC) insecticides chlordane, o,p'-DDT, dieldrin, endosulfan, kepone, methoxychlor, and toxaphene on human HepG2 cytochrome P450 (1A-EROD and 2B-PROD) activities and glutathione (GSH) levels. Cells were exposed for 24 h at high concentrations (1, 5 or 10 mM) and for 48 h at lower concentrations ranging from 0.01 to 1 mM to evaluate dose responses. Our results show that after 48 h all but dieldrin significantly induced both P4501A and 2B. P4502B responses were greater at all exposure concentrations and times. Mixed responses in GSH levels were observed. All OCs except dieldrin and MXC significantly depleted GSH after 24 h. At 48 h, chlordane, endosulfan and toxaphene significantly increased GSH at low levels and decreased GSH at high levels, while kepone and methoxychlor produced significant declines in GSH at all concentrations. These results support findings of OC insecticides inducing CYP1A, 2B in rats, with CYP2B responses more important. GSH levels declined when P4502B activity was significantly elevated and were significantly increased in the absence of significant P450 activity, suggesting that GSH levels influence the catalytic activity of the cytochrome P450s and the cytochrome P450s influence the cell's ability to regulate GSH.

Characterization of the human hepatocellular carcinoma (hepg2) cell line as an in vitro model for cadmium toxicity studies.

Biochemical indicators and in vitro models, if they mimic in vivo responses, offer potentially sensitive tools for inclusion in toxicity assessment programs. The purpose of this study was to determine whether the HepG2 cell line would mimic known in vivo or in vitro (or both) responses of mammalian systems when confronted with cadmium (Cd2+). Uptake and compartmentalization of Cd2+, metallothionein (MT) compartmentalization, and glutathione (GSH) depletion were examined. In addition, several cytotoxic and stress effects, e.g., viability (neutral red [NR] uptake, 3-[4,5-dimethylthiozole-2-yl]-2,5,-biphenyl tetrazolium bromide [MTT] dye conversion, and live/dead [L/D]), membrane damage (lactate dehydrogenase leakage), metabolic activity (adenosine triphosphate levels), and detoxification capabilities (GSH content, cytochrome P4501A1/2 [EROD (ethoxyresorufin-o-deethylase)] activity, and MT induction), were measured in both naive (no previous exposure) and Cd2+ preexposed cells. Cadmium uptake increased during a 24-h period. Metallothionein induction occurred in response to both Cd2+ and ZnCl2; however, Cd2+ was the more potent inducer. Both Cd2+ and MT were localized primarily in the cytoplasmic compartment. All biochemical responses, except EROD, showed concentration- response relationships, after 24-h exposure to Cd2+ (ranges 0-3 ppm [26.7 microM]). Cadmium effects were reduced in preexposed cells, indicating adaptive tolerance or increased resistance had occurred. Twenty-four-hour LC50, dose causing death of 50% of the test subjects, values were 0.97, 0.69, and 0.80 ppm (8.7, 6.2, and 7.2 microM) for naive cells and 1.45, 1.21, and 1.39 ppm (12.9, 10.7, and 12.3 microM) for preexposed cells based on the NR, MTT, and L/D assays, respectively. These data indicate that this carcinoma cell line is a useful in vitro model for cadmium toxicity studies.

A kinetic microassay for glutathione in cells plated on 96-well microtiter plates.

We describe a modified enzymatic, kinetic, glutathione microassay based on the original macroassay described by Tietze and modified by Anderson. It is coupled with the Triton X-100 lysis and an acid extraction that can be performed in 96-well microtiter plates. The microassay can be read in a microplate reader equipped with a standard 405 nm filter. Intracellular glutathione levels are not significantly different when comparing the proposed Triton X-100 lysis and acid extraction method from those found with the cellular homogenization and acid extraction method typically employed. By combining a rapid sample extraction method, which can be done on the microtiter plate, with an assay based on the technology of a microplate reader, we have devised a rapid, reproducible, inexpensive and easy to use GSH microassay that can process several hundred samples daily and will be useful in studies where many replicate samples are required. We have successfully used this method to monitor glutathione status in a human cell line exposed to organochlorine pesticides and cells exposed to naturally occurring mixtures of metals extracted from aquatic sediments.

Nutritional status and energy metabolism of crayfish (Procambarus clarkii, Girard) muscle and hepatopancreas.

1. Food deprivation resulted in significant decreases in muscle carbohydrate, lipid and water content and increased ATP, ADP, AMP and total adenylate levels over the 21-day experimental period. 2. In the hepatopancreas phosphoarginine was significantly higher on day 21 in the starved crayfish. 3. Muscle energy charges remained within optimal (unstressed) ranges, while hepatopancreatic energy charges of food-deprived crayfish fell into suboptimal (stressed) ranges, indicating the necessity of examining organs separately to accurately ascertain metabolic changes in response to stressors.

Energy metabolism in largemouth bass (Micropterus floridanus salmoides) from stressed and non-stressed environments: adaptations in the secondary stress response.

White muscle proteins, carbohydrates, lipids, adenylates, phosphagen and the AEC, (adenylate energy charge) were measured in bass inhabiting stressful and non-stressful environments. Within an environment, in June stressed bass had higher carbohydrates, while non-stressed bass had lower ATP, total adenylates and AEC. Comparisons between environments revealed: non-stressed bass had higher ATP/ADP and AEC's in May, CrP/ATP in June, and protein in July; while stressed bass had higher carbohydrate in June and lipid in July. Other metabolites varied insignificantly. AEC's of both groups were within the optimal range indicating physiological compensation (adaptation) of energy metabolism (secondary stress response) occurred in bass inhabiting the stressful environment.

Adenylate energy charge, arginine phosphate and ATPase activity in juvenile Homarus americanus during the molt cycle.

Neither gill nor hepatopancreas exhibited significant differences in Na+, K+-ATPase activity with molt stage. Hepatopancreatic residual ATPase activity was significantly higher (F = 6.273) in post-molt animals; while gill residual ATPase activity exhibited no significant differences. Muscle AEC did not change with molt stage, but levels of ATP (F = 8.050) and ADP (F = 4.130) were significantly higher in premolt (D3 pleopod stage 5.0-5.5) animals; while levels of arginine phosphate (F = 6.981) were significantly higher in post-molt animals. Arginine phosphate/ATP and ATP/ADP ratios were highest in post-molt animals, but were not statistically significant. Although not significant, changes in Na+, K+-ATPase activity and AEC did suggest alterations in: enzyme activity that correlate with known osmotic compensations occurring during the water uptake and hardening/mineralization processes; and energy metabolism which occur during the molt cycle, respectively.