Possibly the Cr, bound in specific organic residues, is readily available for uptake by J. romerianus. This finding may suggest that this plant holds promise for phytoremediating certain Cr-con-taminated sediments.In Puerto Rico, the U.S. Armed Forces, the National Guard and the Police Department use and store large quantities of ammunitions for their field-range firing maneuvers. Many of the areas used are adjacent to agricultural fields and water resources, which eventually become contaminated with TNT, RDX, or both. Moreover, many sites in Puerto Rico are contaminated with organic com-pounds, including halogenated solvents, aromatic hydrocarbons and metal ions. These compounds are known to be toxic and mutagenic to plants, animals and humans. Therefore, bio-remediation of these wastes in soils and water is very important. Previous studies from abroad have demonstrated that both fungi and bacteria are capable of degrading these compounds. In addition, several plant species have been found capable of absorbing heavy metals from the soil. It is of interest to study the fungal flora predominant in Puerto Rico; few or none are capable of flourishing on media containing these compounds. Since lignin and cellulose-degrading fungi have been reported as potential candidates for the bio-remediation of areas contaminated by munition wastes, this project will place great emphasis on fungal genera within the class Basidiomycetes, which includes several aggressive wood rotters. Aspergillus and Penicillium will also be tested. Chemical compounds and metals such as those mentioned above will be incorporated into culture media, both solid and liquid. Each fungal genus to be tested will be added to the media and incubated,drainage for plants in pots and the extent of growth determined. Growth will be based on fungal dry weights as well as colony diameter in petri plates.

The extent of bio-degradation, if any, will be determined by conventional laboratory analysis . Fungal genera found to be capable of degrading the compounds under study will be tested further, using more sophis-ticated experiments and equipment.Fungal were originally isolated in pure culture using culture media specific for fungal isolation, including potato dextrose agar ; malt extract agar ; and sabouraud agar modified . Fungi such as wood rotters were obtained in pure cultures and preserved on MEA throughout this research. These fungi included: Junhnia straminea, Phlebia chrysocrea, Ganoderma, australe, Tinctosporellus epimiltimus, Programme albocincta, Lactiporus persicinus, Rogodoporus microporus and Antrodierella sp. Other fungi, such as species of Aspergillus and Penicillium, were obtained from contaminated bacterial cultures mixed with heavy metals and preserved on fresh media . The chemicals tested were selenite, arsenite and p-concentrations of 98 mM for selenite and arsenite and 50 mM for p-nitrophenol. There were three different concentrations: 79.0 ppm, 158 ppm and 316 ppm for selenite; 37.5 ppm, 75.0 ppm and 150 ppm for arsenite; and 69.5 ppm, 139 ppm and 278 ppm for p-nitrophe-nol. The effects of selenite, arsenite and p-nitrophenol on growth of the fungi was determined in liquid media mixed with the chemicals. A volume of each chemical corresponding to these concentrations was dispensed into each flask containing 50 ml of the media.The subsurface of central California, including the San Joaquin valley, is highly contaminated with toxic metal oxyanions. The processing of San Joaquin Valley crude oil and agricultural drainage extracted from the subsurface results in the discharge of approximately 2200 kg/year of selenium oxyanions into the San Francisco Bay Area ecosystem. Selenium is toxic to fish, shellfish, waterfowl and small mammals. There is currently no cost-effective technology to treat high-volume selenium-contaminated waste streams. The absence of such technology threatens the environmental quality of San Francisco Bay and other aquatic ecosystems. Certain soil and aquatic bacteria are able to biotrans-form selenium and other toxic metal oxyanions into non-toxic metal precipitates that are immobilized within the bacterial biomass.

Bacillus subtilis is one of these selenium-detoxifying soil bacteria. One goal of this project is to identify and characterize the gene/s involved in hazardous metal detoxification in B. sub-tilis. This should allow us to investigate the mechanism/s of detoxification and provide us with the necessary knowledge to improve the detoxification capacity of B. subtilis through genetic/metabolic engineering and directed evolution. We expect that these findings will also have considerable value in under-standing the biovalence transformation of other toxic metal oxyanions, such as chromate and arsenite. A defined minimal growth medium developed in our laboratory was used for these studies. A genome-wide array technique was used to profile the expression of all of the identified open reading frames in B. subtilis . Total RNA was extracted from cultures of unexposed 168 cells , and selenium-induced 168 cells at three time points: prior to selenite addi-tion at 95 Klett ; two hours after exposure to selenite ; and 45 hours after exposure to selenite when 168 had fully recovered . 33P-labeled cDNAs were prepared from these RNA samples by reverse transcription. The labeled RNA was hybridized to genome-wide arrays obtained from Eurogentec, containing a duplicate set of oligonu-cleotides for all of the B. subtilis ORFs. The expression profile of each gene was determined by the ratio of the digitized intensity values for control and experimental time points.The initial growth arrest of 168 following expo-sure to selenite is primarily due to the formation and incorporation of seleno-cysteine and seleno-methion-ine into proteins. Selenium is very similar in chemical properties to sulfur and is capable of participating in similar biochemical reactions. To investigate the bio-chemical pathways affecting selenite toxicity in B.subtilis, the following sulfur amino acid bio-synthetic mutants, cysA, metC and cysA metC, were constructed. If selenite toxicity is due to the incor-poration of seleno-cysteine and seleno-methionine into proteins, then selenite toxicity should be reduced in cysA and metC single mutants.

These mutants are defective in the early stages of de novo cysteine and methionine biosynthesis. The cysA metC double mutant should com-pletely block de novo synthesis of sulfur amino acids. Addition of cys-teine and/or methionine to the mutant and wild type strains might also physiologically mitigate selenite tox-icity. The results shown in Figure 1 demonstrate that de novo sulfur amino acid bio-synthetic pathways are indeed the primary targets for selenite toxicity.The adaptation to selenite stress observed in 168 is irreversible. Induced B. subtilis no longer undergo growth arrest or cell lysis upon subsequent selenite exposure. We wished to examine whether this induction was permanent or transient. A single induced clonal population was grown for 12, 48 and 70 generations in the absence of selenite stress. These cells were then allowed to sporulate. All of these lineages maintained the induced phenotype. These results indicated that adaptation persisted over a large number of gen-erations in the absence of selenite stress. 3. Genome-wide expression profiling of selenite stress has identified genes involved in general stress response, protein turnover, energy production and operons with no previously known functions. We have employed genome-wide expression profiling techniques to identify genes that are involved in toxic metal adaptation and detoxification processes. Analysis of these data suggests that upon initial expo-sure to selenite,30 litre pot there is an increase the expression of damaged protein turnover and refolding genes . There are also significant increases in the expression of genes involved in DNA recombination and repair suggesting that selenite-induced growth arrest may adversely affect cell cycle events. A number of genes involved in ion efflux and heavy metal binding are overproduced in 168 . Following recovery from selenite stress, genes involved in energy production and components of the electron transport chain are highly induced. This may indicate that selenite detoxification is energetically intensive. The expression of thioredoxin and thioredoxin reduc-tase increased several fold by T3. These genes have been shown to catalyze the in vivo reduction of selen-ite to nontoxic elemental selenium. Previous proteomic analysis has also implicated these proteins in the man-agement of selenite stress. We have identified a number of operons with no pre-viously known function that could be involved in hazardous metal adaptation and/or detoxification. For example, yxlA through yxlJ are found within an operon that also includes a sigma factor of unknown function, sigY . The expression of the genes in this operon increased up to 28-fold during the recovery from selenite stress . Organochlorines and polycyclic aromatic hydrocarbons are ubiquitous environmental contaminants that are toxic and suspected human carcinogens. Traditional assessment of human exposures to these organic toxins and subsequent biological response relies primarily on high-dose and short-term animal experiments. A major uncertainty inherent to this approach is the extrapolation from the high-dose animal experiments to the low-dose and long-term human exposures. To overcome this uncertainty, we recently have directed our research towards exploring the use of synchrotron radiation-based Fourier transform infrared spectromicroscopy for identifying chemical changes in cellular nucleic acids and proteins as a result of OC and PAH exposures.

To date, the primary research objective has been to identify SR FTIR spectroscopic signals of human cell culture systems that are indicative of low-dose exposures to OCs and PAHs and could be used as biomarkers. SR FTIR spectromicroscopy is used because this is a sensitive and nondestructive technique capable of providing direct biochemical information at molecular levels. The fine spatial resolution of 5-10 microns and strong signal-to-noise levels of SR FTIR spectromicroscopy allow detection of the subtle changes in intracellular biochemical processes as the cells are exposed to environmental stimuli. The biomarker considered for OC and PAH exposures is the induction of the cytochrome P4501A1 CYP1A1 gene expression and the increase in the asso-ciated enzyme activity. It is well established that CYP1A1 transcript levels increase in response to expo-sure to OCs and PAHs through their binding to the Ah receptors. HepG2 cells were used as model human epithelial cells that can metabolize PAHs; 2,3,7,8-tetrachlorodibenzo-p-dioxin modeled OCs; BaP modeled PAHs and coal tars modeled mixtures of PAHs. HepG2 cells were maintained in Eagle’s Minimum Essential Medium with non-essential amino acids and Earle’s BSS supplemented with 10% fetal calf serum, 1 mM L-glutamine, 10 mM Hepes and antibiotics. Cells were sub-cultured and then treated for 2-20 hours with TCDD, BaP and coal tars at environmentally relevant concentrations. The effect of TCDD was monitored and mapped by the SR FTIR spectromicroscopy technique in the mid-IR region . SR FTIR signals that are specific to the intracellular response after their exposure to these organic toxins were identified. To validate the SR FTIR spectromi-croscopy technique, results from the TCDD experiments were compared with the CYP1A1 tran-script levels measured by the widely accepted yet more time-consuming reverse transcription-poly-merase chain reaction .Dimensionless SR FTIR spectra were recorded at the proximity of a cell nucleus of HepG2 cells that were exposed to TCDD of different concentrations for 20 hours. There are consider-able differences in the SR FTIR spectra associated with the CYP1A1 gene expression and the increase in the associated enzyme activity, with one difference being the increased absorption of the vibration band 1180 – 1160 cm-1, centered at ~1170 cm-1. Here, the normalized absorbance intensity for individual cells increased from 0.007 to 0.21 when the TCDD concentration increased from 10-11 to 10-9 M . The normalized absorbance intensity at ~1170 cm-1 for individual control cells was 0.005, a 42-fold increase in the absorbance intensity. This systematic spectral change might be related to the alteration in the DNA base structure and will be the subject of future investigation. A comparison of the dose response described above with that obtained using the RT-PCR technique is shown in Figure 1b. The solid line was the least-squares fit to the data. The excellent agreement for measurements from the two methods indicated that the fast and direct SR FTIR spectromi-croscopy technique was indeed comparable to the more time-consuming and widely accepted RT-PCR technique that specifically measures increases in the CY1A1 transcript levels. The SR FTIR spectra recorded at the proximity of a cell nucleus of HepG2 cells that have been exposed to BaP and coal tars at environmentally relevant concentrations showed similar spectral characteristics at ~1170 cm-1. The recorded dose-response behavior was also similar to those reported in literature. The agreement between the SR FTIR spectromicro-scopic data for dioxin exposures and the RT-PCR results, and the agreement between the PAH measurements and those reported in the literature indicate that the intracellular biological responses to low-dose exposures to these organic toxins are well represented by our specific spectral changes. These changes are associ-ated with CYP1A1 gene expression and the increase in the associated enzyme activity with different types of damage.