Multiple xenobiotic-inducible P450s are involved in alkoxycoumarin and alkoxyresorufin metabolism in higher plants

O-Dealkylation of two series of fluorescent 7-alkoxy-coumarins and 7-alkoxyphenoxazones by plant cytochrome P450s was investigated in Helianthus tuberosus tuber tissues treated with prototype P450 inducers, environmental pollutants or agrochemicals. Methoxy-, ethoxy-, propoxy-and butoxycoumarins and methoxy- and ethoxyresorufins were metabolized by fplant microsomes. Dealkylation of pentoxy- and benzyloxyresorufins was not detected. All dealkylating activities were enhanced by aging plant tissues in the presence of xenobiotics, in some cases up to 20-fold relative to the activities detected in control tissues. Increases in total P450 in the same tissues never exceeded 3-fold. The isozymes induced by prototype P450 inducers clearly differed from those in mammalian liver. That multiple P450s with overlapping substrate specificities were involved in the metabolism of both alkoxycoumarins and alkoxyresorufins was demonstrated by (1) the differential induction of the activities in response to exposure to xenobiotics, (2) the differential inhibition of the activities by clotrimazole, paclobutrazole and tetcyclacis in aminopyrine and benzo(a)pyrene-treated tissues, and(3) the selective inhibition observed with antibodies raised against purified ethoxycoumarin deethylase fractions. Our results suggest that the measurement of the dealkylation of such fluorescent substrates in plants might be useful to monitor environmental pollution.     

Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia

Toxic aromatic pollutants, concentrated in industrial wastes and contaminated sites, can potentially be eliminated by low cost bioremediation systems. Most commonly, the goal of these treatment systems is directed at providing optimum environmental conditions for the mineralization of the pollutants by naturally occurring microflora. Electrophilic aromatic pollutants with multiple chloro, nitro and azo groups have proven to be persistent to biodegradation by aerobic bacteria. These compounds are readily reduced by anaerobic consortia to lower chlorinated aromatics or aromatic amines but are not mineralized further. The reduction increases the susceptibility of the aromatic molecule for oxygenolytic attack. Sequencing anaerobic and aerobic biotreatment steps provide enhanced mineralization of many electrophilic aromatic pollutants. The combined activity of anaerobic and aerobic bacteria can also be obtained in a single treatment step if the bacteria are immobilized in particulate matrices (e.g. biofilm, soil aggregate, etc.). Due to the rapid uptake of oxygen by aerobes and facultative bacteria compared to the slow diffusion of oxygen, oxygen penetration into active biofilms seldom exceeds several hundred micrometers. The anaerobic microniches established inside the biofilms can be applied to the reduction of electron withdrawing functional groups in order to prepare recalcitrant aromatic compounds for further mineralization in the aerobic outer layer of the biofilm.Aside from mineralization, polyhydroxylated and chlorinated phenols as well as nitroaromatics and aromatic amines are susceptible to polymerization in aerobic environments. Consequently, an alternative approach for bioremediation systems can be directed towards incorporating these aromatic pollutants into detoxified humic-like substances. The activation of aromatic pollutants for polymerization can potentially be encouraged by an anaerobic pretreatment step prior to oxidation. Anaerobic bacteria can modify aromatic pollutants by demethylating methoxy groups and reducing nitro groups. The resulting phenols and aromatic amines are readily polymerized in a subsequent aerobic step.     

Abbau von 14C-, 3H- und 36Cl-markiertem γ-Hexachlorcyclohexan durch anaerobe Bodenmikroorganismen

Zusammenfassung Durch eine anaerobe Mischflora aus Ackerboden wurde -Hexachlorcyclohexan (-HCH) in 4–5 Tagen zu 90% abgebaut. Dabei erfolgte eine schnelle Abspaltung des Chlors in Form von Chloridionen und danach eine Freisetzung des C- und H-Anteiles in Form flüchtiger Verbindungen, in denen kein Chlor und auch kein CO₂ nachzuweisen war.Die Verwendung von ¹⁴C/³H- und ³⁶Cl/³H-doppelmarkiertem -HCH zeigte, daß die Cl- und H-Abspaltung nicht im Verhältnis von 1:1 erfolgte, sondern mehr Cl als H abgespalten wurde. Die flüchtigen Verbindungen enthielten andererseits höhere ¹⁴C- als ³H-Anteile. Gaschromatographische Untersuchungen zeigten ebenfalls eine rasche Verminderung des -HCH und die Bildung verschiedener Metabolite. Es wurde jedoch kein -Pentachlorcyclohexen nachgewiesen. Bei steigenden O₂-Gehalten in der Gasphase verminderte sich der -HCH-Abbau. Jedoch fanden auch noch bei 5% O₂ Chlorabspaltung und die Freisetzung flüchtiger Metabolite statt.-HCH wurde ebenfalls, jedoch langsamer, durch die anaerobe Mischflora abgebaut. Auch hier wurde Chlorid abgespalten, und es traten ebenfalls flüchtige Verbindungen auf, die kein Chlor enthielten.

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Degradation of ¹⁴C-, ³H- and ³⁶Cl-labelled -hexachlorocyclohexane by anaerobic soil microorganisms

Up to 90% of the -Hexachlorocyclohexane (-HCH) applied to an anaerobic mixed bacterial flora enriched from an arable soil were degraded within 4–5 days. Degradation resulted in a rapid release of chloride and in formation of chlorine-free volatile metabolites. CO₂ formation from the molecule was not detected.Investigations with ¹⁴C/³H- and ³⁶Cl/³H double-labelled -HCH indicated that the release of Cl and H did not occur in the ratio of 1:1. More Cl than H was split off. The volatile compounds contained more ¹⁴C than ³H. Gas chromatographic studies also showed the rapid decrease of -HCH and the formation of several metabolites. -Pentachlorocyclohexene was not detected. Increasing O₂-contents in the gas phase of cultures resulted in decreases of the compound's degradation. Release of chloride and of volatile metabolites were observed with O₂ contents in the gas phase up to 5%.-HCH was also, but more slowly as with -HCH, degraded by the anaerobic mixed flora. Chloride was released and volatile, chlorine-free metabolites were found.

     

Genetic polymorphisms in GSTM1, GSTT1, GSTP1, GSTM3 and the susceptibility to gallbladder cancer in North India

Abstract

The glutathione S-transferase (GSTs) are polymorphic supergene family of detoxification enzymes that are involved in the metabolism of numerous potential carcinogens. Several allelic variants of polymorphic GSTs show impaired enzyme activity and are suspected to increase the susceptibility to various cancers. To find out the association of GST variants with risk of gallbladder cancer, the distribution of polymorphisms in the GST family of genes (GSTT1, GSTM1, GSTP1, and GSTM3) were studied in 106 cancer patients and 201 healthy controls. Genotypes were analysed by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP). The frequencies of GSTM1 null and GSTM3*BB genotypes did not differ between patients and controls. The overall frequency of GSTT1 null was lower in cases as compared with controls (p=0.003, Odds ratio (OR)?=?0.2, 95% confidence interval (CI), 0.1–0.6). After sex stratification, the GSTT1 null frequency was reduced only in female patients (p=0.008, OR?=?0.2, 95% CI?=?0.1–0.6). However, the GSTP1, ile/val genotype and the val allele were significantly higher in cases than controls (p=0.013, OR?=?1.9, 95% CI?=?1.1–3.1; p=0.027, OR?=?1.5, 95% CI?=?1.0–2.1), respectively. To study gene–gene interactions, a combined risk of gallbladder cancer due to ile/val or val/val were calculated in combination with null alleles of GSTM1 and GSTT1 or the *B allele of GSTM3, but there was no enhancement of risk. Gallstones were present in 57.5% of patients with gallbladder cancer, but there were no significant differences between allelic/genotype frequencies of the studied GST genes polymorphisms between patients with or without gallstones. To best of our knowledge, this is the first paper showing ile/val genotypes and val allele of GSTP1 to be associated with higher risk of gallbladder cancer.     

Phytoremediation—A Novel and Promising Approach for Environmental Clean-up

ABSTRACT

Phytoremediation is an eco friendly approach for remediation of contaminated soil and water using plants. Phytoremediation is comprised of two components, one by the root colonizing microbes and the other by plants themselves, which degrade the toxic compounds to further non-toxic metabolites. Various compounds, viz. organic compounds, xenobiotics, pesticides and heavy metals, are among the contaminants that can be effectively remediated by plants. Plant cell cultures, hairy roots and algae have been studied for their ability to degrade a number of contaminants. They exhibit various enzymatic activities for degradation of xenobiotics, viz. dehalogenation, denitrification leading to breakdown of complex compounds to simple and non-toxic products. Plants and algae also have the ability to hyper accumulate various heavy metals by the action of phytochelatins and metallothioneins forming complexes with heavy metals and translocate them into vacuoles. Molecular cloning and expression of heavy metal accumulator genes and xenobiotic degrading enzyme coding genes resulted in enhanced remediation rates, which will be helpful in making the process for large-scale application to remediate vast areas of contaminated soils. A few companies worldwide are also working on this aspect of bioremediation, mainly by transgenic plants to replace expensive physical or chemical remediation techniques. Selection and testing multiple hyperaccumulator plants, protein engineering of phytochelatin and membrane transporter genes and their expression would enhance the rate of phytoremediation, making this process a successful one for bioremediation of environmental contamination. Recent years have seen major investments in the R&D, which have also resulted in competition of filing patents by several companies for economic gains. The details of science & technology related to phytoremediation have been discussed with a focus on future trends and prospects of global relevance.     

Microbiological and Biotechnological Aspects of Metabolism of Carbamates and Organophosphates

Abstract

Several carbamate and organophosphate compounds are used to control a wide variety of insect pests, weeds, and disease-transmitting vectors. These chemicals were introduced to replace the recalcitrant and hazardous chlorinated pesticides. Although newly introduced pesticides were considered to be biodegradable, some of them are highly toxic and their residues are found in certain environments. In addition, degradation of some of the carbamates generates metabolites that are also toxic. In general, hydrolysis of the carbamate and organophosphates yields less toxic metabolites compared with the metabolites produced from oxidation. Although microorganisms capable of degrading many of these pesticides have been isolated, knowledge about the biochemical pathways and respective genes involved in the degradation is sparse. Recently, a great deal of interest in the mechanisms of biodegradation of carbamate and organophosphate compounds has been shown because (1) an efficient mineralization of the pesticides used for insect control could eliminate the problems of environmental pollution, (2) a balance between degradation and efficacy of pesticides could result in safer application and effective insect control, and (3) knowledge about the mechanisms of biodegradation could help to deal with situations leading to the generation of toxic metabolites and bioremediation of polluted environments. In addition, advances in genetic engineering and biotechnology offer great potential to exploit the degradative properties of microorganisms in order to develop bioremediation strategies and novel applications such as development of economic plants tolerant to herbicides. In this review, recent advances in the biochemical and genetic aspects of microbial degradation of carbamate and organophosphates are discussed and areas in need of further investigation identified.     

Growth of Chlorinated Solvent‐Degrading Consortia in Fed‐Batch Bioreactors and Development of a Double‐Substrate High‐Performing Microbial Inoculum

The long‐term growth process of two microbial consortia effective in the aerobic cometabolic biodegradation of a mixture of 6‐chlorinated aliphatic hydrocarbons (CAHs), the effectiveness of these consortia as inocula for the bio‐augmentation of different types of microcosms and the development of a double‐substrate, high‐performing consortium is presented. The propane‐utilizing consortium generally proved to be the most effective one, being able to biodegrade vinyl chloride, cis‐ and trans‐1,2‐dichloroethylene, trichloroethylene, 1,1,2‐trichloroethane and 1,1,2,2‐tetrachloroethane at all the CAH concentrations tested (0–4 μM). Both consortia maintained unaltered CAH degradation capacities over a 300‐day growth period in the absence of the CAHs and were effective in inducing the rapid onset of CAH depletion upon inoculation in slurry microcosms set up with five types of aquifer materials. A consortium supplied with both methane and propane combined the best degradation capacities of the two single‐substrate consortia, and maintained stable performances for 150 days under slurry conditions. The degree of conversion of the organic Cl to chloride ions was equal to 90 %.     

Accelerated development of uroporphyria in mice heterozygous for a deletion at the uroporphyrinogen decarboxylase locus

Three weeks after a single dose of iron-dextran and Aroclor 1254, mice maintained continuously on delta-aminolevulinic acid supplemented drinking water showed significantly elevated levels of hepatic uroporphyrin and depressed (25% of normal) uroporphyrinogen decarboxylase (URO-D) activity. Depressed URO-D activity was paralleled by the ability of heat denatured cytosol to inhibit rhURO-D activity. Mice heterozygous for a targeted disruption at the URO-D locus (URO-D+/-) exhibited half the URO-D activity of homozygous controls prior to treatment. After treatment, these animals showed URO-D activity and rhURO-D inhibitory activity comparable to similarly treated wild type (URO-D +/+) mice but with significantly greater uroporphyrin accumulation. With only 10 days of treatment, URO-D +/- but not URO-D +/+ mice showed changes similar in magnitude to those seen after 21 days. Prior to treatment, URO-D genotype did not influence overall hepatic P450 concentration in either sex and there was no significant difference between sexes. The treatment regimen significantly elevated P450 in animals of either URO-D genotype and in both sexes, although the induction response at the 10-day point was attenuated in URO-D +/- mice. From differences in the CO absorbance maximum, and by P450 activity analysis, this attenuated induction response resulted from an attenuation of the CYP2B not the CYP1A induction.