The capabilities of SR FTIR spectromi-croscopy for the direct detection of intracellular biochemical responses to exposures to dilute concentrations of OCs and PAHs will have significant impacts in future research methodology of environmental toxicology.Bio-assays used in aquatic toxicology have taken a prominent position among analytical tests for identifying and measuring environmental hazards. Such bio-assays have been developed for testing a variety of organic and inorganic chemicals, as well as effluents, surface waters and sediment samples for acute and chronic toxicity. Many bio-assays using higher organ-isms such as fish, protozoa and algae have been executed, but are labor- and equipment-intensive, costly and complex. More importantly, these aquatic bio-assays do not provide quantitative information on the impact of pollutants on biological treatment systems. In the view of national and international regulations, regulatory agencies are also supporting the development of new toxicity screening procedures that are sensitive, inexpensive and easier to perform. The use of bacterial in vitro assays such as the Microtox Assay has become an attractive alternative to traditional fish and invertebrate methods for toxicological screening. These new assays have been developed to assess the toxicity of various environmental agents, validated and recognized by several standards organizations. The purpose of this study was to apply selected microbial test protocols to assessing the toxicity of hazardous metals such as cadmium and lead. These metals have been reported to pose a high level of hazard to ecological and human health.A Microtox assay was carried out to measure the relative acute toxicity of metal producing data for the calculation of lead concentration effecting 50% reduction in light output . For each test run, two controls without lead,pot blueberries eight sample/lead dilutions and two replicates were done. Tests were carried out on various percentages of the original lead concentration . The sensitivity of the strain of bio-luminescent bacteria was tested for quality control purposes. Growth and oxygen uptake experiments were performed following previously described protocols.

Descriptive statistics were applied to calculate the means+SD of all data sets associated with specific metal concentrations. Specific growth and oxygen depletion rates were computed as slopes of graphical representations of raw data versus times. The toxic end-points expressed as 50% growth inhibition concentration or as 50% oxygen depletion concentration were next derived from graphical presentations of these specific rates versus metal concentrations. Activity quotients were calculated to determine the degree of toxicity associated with lead exposure. Linear regression analysis was performed to determine the relationship between lead concentrations and the times required for 50% reduction in oxygen uptake .Bioluminescence was used as an endpoint for measuring the effect of Cd and Pb to Vibrio fischeri. For both Cd and Pb, a strong dose-response relationship was determined. The concentrations of Cd and Pb effecting 50% reduction in bioluminescence were computed to be 0.79+0.12 mg/L and 0.34+0.03 mg/L, respectively; indicating that Pb was more toxic than Cd. A strong dose-response relationship was also found in the tests with the mixed population of microorganisms. Figure 1 shows the growth patterns obtained from exposure to lead of the mixed population of microorganisms. Data presented in this figure show an overall increase in bacterial growth with the increase in holding/incubation time. These data also show significant reductions in maximum growths with increasing concentrations of lead. EC50 values were computed to be 4.50+0.04 mg/L, and 3.50+0.02 mg/L for Cd and Pb, respectively. Figure 2 presents the dissolved oxygen uptake rates by the mixed population of microorganisms exposed to various concentrations of lead. In general, curves presented in this figure indicated that individual rates of oxygen uptake decreased as lead concentrations increased. The mean values of EC50 were 5.00+0.42 mg/L for Cd, and 3.80+0.04 mg/L for Pb. These data indicated that the mixed population of microorganisms was about 10 times less sensitive to lead toxicity than the marine bacterium, Vibrio fischeri. Data also showed a strong correlation between TD50s and lead concentrations, indicating a time-response relationship with regard to lead toxicity. A similar result was obtained in experiments with cadmium. Data obtained from this research clearly point out the significance of using microbiological systems for acute toxicity testing in aquatic toxicology. Bio-assays employed in the present investigation fulfilled the requirement criteria of fast toxicity screening based on their simplicity, speed, cost effectiveness and the fact that bacteria grow rapidly, represent a low trophic level, and thus provide sensitive early warning data of environmental impacts at higher trophic levels.

Of the three bio-systems evaluated, the Microtox was the most sensitive; yielding an EC50s that was only about one tenth of values recorded in batch cultures . Although batch systems were time-consuming , and relatively less sensitive than the Microtox, they provided valuable information on the toxic effects of lead on microbial growth and respiration. Also, they were easier to perform, and required less expensive equipment compared to the high cost of the Microtox analyzer. Trinitrotoluene is a munitions chemical that was produced and used on an enormous scale during World Wars I and II in shells, bombs, grenades, demo-lition explosives and propellant compositions. 2,4-Dinitrotoluene , and 2,6-Dinitrotoluene , on the other hand, are used in the manufacture of dyes, in munitions as smokeless propellant powders, and as gelatinizing and plasticizing agents in both commercial and military explosive compositions. Both 2,4-DNT, and 2,6-DNT are produced through denitration of toluene with nitric acid in the presence of concentrated sulfuric acid. Small amounts of DNT isomers also occur as byproducts in the production of TNT. Significant amounts of TNT- and DNT-contain-ing waste waters arising from their preparation and production at Army ammunition plants have been identified in soils, surface water and ground water after leaching from disposal sites. Exposure to TNT and DNTs has been associated with numerous health effects. However, limited scientific information is available regarding the environmental fate, ecotoxicity and health effects of these nitroaromatic compounds. We have performed the Microtox, Mutatox and CAT-Tox assays to determine the acute toxicity, genotoxicity and molecular mechanisms by which these munitions chemicals exert their toxicity. Acute and genotoxicity tests were carried out, using a Microtox/Mutatox Model 500 Toxicity Analyzer System. The Microtox procedure measured the relative acute toxicity of lead, producing data for the calcula-tion of lead concentration effecting 50% reduction in light output . For each test run, two controls without lead, eight samples/chemical dilutions and two replicates were done. The Mutatox Assay was conducted according to the standard test protocol. Nonglowing or dark mutant strains of luminescent bacteria were exposed to the test substance , and the amount of light emitted was measured with the Mutatox Analyzer. The sample-induced reversion from nonglowing to lumi-nescent phenotype was used to indicate the genotoxicity of the sample.

Prepared samples were mixed and preincubated in a water bath at 35 ± 0.5°C for 45 minutes. After preincubation, samples were incubated at 27 ± 5°C for 16, 20 and 24 hours, and the potential genotoxic response of the luminescent bacte-ria was determined at each time period by measuring the light intensity of each cuvette using the Mutatox Model 500 Analyzer. The positive response was defined as the light output of at least two times the light intensity of the reagent control blank. The mammalian Gene Profile Assay was performed for measuring differential gene expression in the human liver hepatoma cell line, HepG2. Thirteen recombinant cell lines and the parental HepG2 Cell line were plated over two 96-well microplates. The cell lines were dosed at five TNT concentrations and incubated at 37°C, 5% CO2, for 48 hours. After the incubation period,square plastic plant pots the total protein was measured by the Bradford method, at 600 nm using a microplate reader. A standard sandwich ELISA was performed and in the final step horseradish peroxidase catalyzed a color change reaction that was measured at 405 nm. The parental HepG2 cell line was dosed in the same manner as the recombinant cell lines, and was used to perform a MTT-based cellular viability assay at 550 nm.Polycyclic aromatic hydrocarbons are a family of compounds that includes some potent car-cinogens that are ubiquitous in the environment. The major metabolic pathway for ingested or inhaled PAHs to water-soluble derivatives is oxidative activation by cytochrome P4501A1 followed by detoxification by phase II enzymes like glutathione S-transferases, especially GSTM1. Interindividual variation in PAH metabolism exists due to genetic polymorphisms in the genes coding for these enzymes. The GSTM1 gene is frequently deleted in individuals, resulting in reduced detoxification. Several single-base changes have been identified in the CYP1A1 gene that appear to result in increased susceptibility to various cancers in these individuals. Because PAHs present a threat to human health, human exposure to PAHs has to be monitored in occu-pational settings. While PAHs consist of hundreds of different aromatic compounds, pyrene is typically present in all of these mixtures. Pyrene is metabolized primarily to 1-hydroxypyrene and detoxified as 1OHP sulfate or glucuronide conjugate and excreted via the urine. Through simple enzymatic methods, these conjugating molecules can be cleaved. Therefore urinary 1OHP is the most commonly used biomarker of exposure to PAHs. Recently, a number of investigators have reported differences in the quantity of urinary PAH metabolites in individuals with poly-morphisms or variations in key enzymes involved in the metabolism of xenobiotics. These findings suggest the need for clarification of the effects of polymor-phisms on the metabolism of pyrene. To investigate the role of these polymorphisms, we have undertaken a study to measure 1OHP levels.Genetic polymorphisms: Peripheral blood lympho-cytes are isolated from the blood samples using Histoprep density separation media . DNA is extracted from the PBLs using standard phenol chloro-form extraction methods. Polymorphisms are analyzed by published procedures: For CYP1A1, the procedure described by Cascorbi et al. is followed.

For identification of CYP1A1 M1, a 899 bp fragment is amplified, then digested with MspI which cuts the variant fragment into a 693 and 206 bp fragment. For the identification of the CYP1A1 M2 polymorphism, a 204 bp DNA fragment is amplified, then subjected to digestion with BsrDI, which cuts the wildtype into a 149 and 55 bp fragment. Restriction enzyme digested PCR products are separated by agarose gel elec-trophoresis. GST analysis is performed using a multiplex PCR that co-amplifies the GSTM1 and GSTT1 genes . An actin DNA frag-ment is co-amplified as an internal control. The absence of a GSTM1 or GSTT1 band in the presence of the actin band indicates a GST gene deletion. Analysis of 1OHP: Methods for analysis of 1OHP follow the protocol by Whiton et al. , which involves overnight enzymatic digestion of all conju-gated forms of pyrene in a 25 ml urine sample, organic extraction of 1OHP, and reverse phase HPLC analysis and quantitation of the 1OHP peak.Inorganic pyrophosphate is an intermediate compound generated by a wide range of metabolic processes, including biosynthesis of various macromolecules such as proteins, DNA, RNA, and polysaccharides. Being a high-energy phosphate compound, PPi can serve as a phosphate donor and energy source, but it can, at high levels, become inhibitory to cellular metabolism. To maintain an optimal PPi level in the cytoplasm, timely degradation of excessive PPi is carried out by two major types of enzymes: soluble inorganic pyrophosphatases and proton-translocating membrane-bound pyrophosphatases. The importance of maintaining an optimal cellular PPi level has been demonstrated in several different organisms. Genetic mutations that lead to the absence of sPPase activity affects cell proliferation in Escherichia coli. In yeast, inorganic pyrophosphatase is indispensable for cell viability because loss of its function results in cell cycle arrest and autophagic cell death associated with impaired NAD+ depletion. In Arabidopsis, a tonoplast-localized proton-pumping pyrophosphatase AVP1 was shown to be the key enzyme for cytosolic PPi metabolism in different cell types of various plants. This enzyme activity has been correlated with the important function that AVP1 plays in many physiological processes. Arabidopsis fugu5 mutants lacking functional AVP1 show elevated levels of cytosolic PPi and display heterotrophic growth defects resulting from the inhibition of gluconeogenesis. This important role in controlling PPi level in plant cells is reinforced by a recent study showing that higher-order mutants defective in both tonoplast and cytosolic pyrophosphatases display much severe phenotypes including plant dwarfism, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Moreover, the tonoplast-localized H+ -PPase AVP1 appears to be a predominant contributor to the regulation of cellular PPi levels because the quadruple knockout mutant lacking cytosolic PPase isoforms ppa1 ppa2 ppa4 ppa5 showed no obvious phenotypes.