Mixed-function oxidase induction as a test for the biological monitoring of water: limitations and prospects]

The induction in fish liver of some enzyme activities, and typically of microsomal mixed-function oxidases (MFO), provides the earliest biological warning signal of exposure to pollutants. Our studies provided evidence that the basal levels of cytochrome P-450 and specific MFO activities, such as arylhydrocarbon hydroxylase (AHH) and ethoxyresorufin deethylase (EROD), were strongly influenced by the diet in freshwater fish (Oncorhynchus mykiss). The response of fish liver to a known enzyme inducer, i.e., beta-naphthoflavone, was also affected by the diet, which therefore should be carefully controlled in laboratory studies. Under field conditions MFO activities were significantly enhanced in the liver of O. mykiss kept in polluted river water as well as in the liver of the seawater fish Diplodus annularis collected from a polluted harbour area, as compared to specimens of the same species collected from an unpolluted reference area.     

Interrelationships between bioaccumulation of organic trace pollutants (PCBs,organochlorine pesticides and PAHs), and MFO-induction in fish

• 1.1. Hydrophobic compounds that are more easily biotransformed (e.g. PAHs) generally show less bioaccumulation in fish than the more persistent PCBs and DDTs.
• 2.2. In lake Nieuwe Meer, with the highest levels of organic micropollutants, the hepatic MFO activity was elevated in three fish species. This indicates that MFO activity might be a sensitive indicator for organic micropollutants in the aquatic environment.
• 3.3. Activities of the 3-MC type inducible isozymes were most pronounced in all three fish species studied. Though further research is required, indications for the existence of a PB-type inducible enzyme system have been demonstrated for two fish species, i.e. pike and eel.
• 4.4. Fish liver enlargement as a consequence of MFO induction could not be demonstrated in fish from the most polluted lake.
• 5.5. In fish, a correlation was observed between PCB or OCP tissue concentrations on the one hand, and hepatic MFO activities on the other. The low PAH fish/sediment ratios are further reduced when MFO systems are induced due to organic micropollution. The interrelationship between bioaccumulation and enzyme induction demonstrates the importance of an integrated study of these phenomena in field research.

     

The periphyton as a multimetric bioindicator for assessing the impact of land use on rivers: an overview of the Ardières-Morcille experimental watershed (France)

Developing new biological indicators for monitoring toxic substances is a major environmental challenge. Intensive agricultural areas are generally pesticide-dependent and generate water pollution due to transfer of pesticide residues through spray-drift, run-off and leaching. The ecological effects of these pollutants in aquatic ecosystems are broad-ranging owing to the variety of substances present (herbicides, fungicides, insecticides, etc.). Biofilms (or periphyton) are considered to be early warning systems for contamination detection and their ability to reveal effects of pollutants led researchers to propose a variety of methods to detect and assess the impact of pesticides. The present article sought to provide new insights into the ecological significance of biofilm microbial communities and to discuss their bioindication potential for water quality and land use by reporting on 4 years of research performed on the French Ardières-Morcille experimental watershed (AMEW). Various biological indicators have been applied during several surveys on AMEW, allowing the characterisation of (i) the structure and diversity of biofilm communities [community level finger printing (CLFP) such as PCR–DGGE and pigment classes], (ii) functions associated with biofilm [community level physiological profiles (CLPP) such as extracellular enzymes, pesticides biodegradation or carbon sources biodegradation] and (iii) biofilm tolerance assessment (pollution-induced community tolerance, PICT) of the main contaminant in the AMEW (copper and diuron). Approaches based on CLFPs and PICT were consistent with each other and indicated the upstream–downstream impact due to the increasing land use by vineyards and the adaptation of algal and bacterial communities to the pollution gradient. CLPPs gave a contrasted bioindication because some parameters (most of the tested extracellular enzymes activities) did not detect a pollution gradient. Such CLPPs, CLFPs and PICT methods applied to biofilm could constitute the basis for a relevant in situ assessment both for chemical effects and aquatic ecosystem resilience.     

Short review of selected fish biomarkers of xenobiotic exposure with an example using fish hepatic mixed-function oxidase

Abstract One group of biological tools that are useful for monitoring exposure to xenobiotics (and hence water quality) have been collectively referred to as biomarkers and are denned in this paper as any biochemical, histological and/or physiological alterations or manifestations of stress. Biomarkers within an aquatic toxicological context generally represent biological responses of individual organisms to xenobiotic exposure (i. e. responses at the whole organism level of biological organization). These include among others, enzyme alterations, bile metabolites, RNA/DNA ratio, adenylate energy charge, skeletal abnormalities, immune dysfunction, behavioural changes and histopathological lesions. Biomarkers can act as effective early warning sentinels to ensure the protection of the integrity of whole ecosystems, including freshwater and marine ecosystems. This paper briefly reviews a selection of fish biomarkers of xenobiotic chemical exposure and discusses their respective strengths and limitations for use in biomonitoring. An example of the application of fish mixed-function oxidase (MFO) and cytochrome P-450 as biomarkers of chemical exposure in Port Phillip Bay is provided. It is concluded that judicious application of biomarkers such as MFO in association with an understanding of the underlying causal mechanisms of induction and toxicity, will contribute to the successful prediction of biological effects of xenobiotic exposure on fish population health.     

Responses of mixed-function oxygenase and antioxidase enzyme system of Mytilus sp. to organic pollution.

1. Mixed-function oxidase (MFO) system components (cytochrome P-450, "418-peak", cytochrome b5 and NADPH-cytochrome c(P-450) reductase) and inducible antioxidant enzymes (catalase, superoxide dismutase (SOD), glutathione peroxidase (GPX) and DT-diaphorase) has been determined in digestive glands of mussels (Mytilus galloprovincialis) collected from three Mediterranean coastal locations, exhibiting an organic pollution gradient. 2. Cytochrome P-450, the "418-peak", catalase and SOD showed a good correlation with whole body tissue PAHs and, to a lower extent, with PCBs. 3. Microsomal NADPH-dependent DT-diaphorase, but not the NADH-dependent microsomal enzyme or the cytosolic DT-diaphorases, was indicated to increase with pollution exposure. 4. The application of such measurements to environmental monitoring is discussed. Given the magnitude of differences observed, and the state of knowledge on enzyme function and mechanisms of toxicity, a multiparameter approach is considered to offer current and future potential for detecting the impact of organic pollution on bivalve molluscs.     

滩涂底栖动物有机污染生态学研究进展

底栖动物由于对有机污染物具有较强的吸收能力,再加上其移动能力较差、生活方式比较固定,而被广泛运用于滩涂有机污染的研究.目前这些研究主要集中在如下几个方面：（1）有机污染物在底栖动物体内的分布特征及在底栖食物链中的动力学研究;（2）底栖动物对有机污染物的生理响应研究;（3）污染物对底栖动物群落组成和结构影响研究;（4）底栖动物在滩涂有机污染检测中的应用研究.研究结果表明：滩涂底栖动物对有机污染物的累积具有选择性和季节波动性;有机污染物可以在底栖食物链中传递;底栖动物体内的有机污染物成分和含量可以有效地指示其生存环境的有机污染状况;底栖动物的混合功能氧化酶和抗氧化酶系统对体内有机污染物的累积产生积极的响应;有机污染物对底栖动物的免疫系统造成不利影响,并对遗传物质造成破坏;有机污染对底栖动物的群落组成和结构具有显著的影响.     

Can biosensors help to protect drinking water?

A large proportion of drinking water is abstracted for treatment from lowland rivers—about 30% in the UK—and this water is at particular risk from sudden and poisonous industrial or agricultural pollution. To cover the range of potential pollutants it may be possible to use biosensors as broadband monitors for toxins. The underlying assumption is that some biological processes, when challenged with a toxin, will be affected in a way analogous to that of man, and that therefore on-line scrutiny of such processes will provide early warning of substances liable to be detrimental to human health. Suitable processes for study might involve multi-cellular organisms, whole cells or enzymes. To date, most practical work has concentrated on fish, but enzymes and single cells hold out the promise of quicker response and, possibly, easier maintenance.     

Interrelationships between bioaccumulation of organic trace pollutants (PCBs, organochlorine pesticides and PAHs), and MFO-induction in fish.

1. Hydrophobic compounds that are more easily biotransformed (e.g. PAHs) generally show less bioaccumulation in fish than the more persistent PCBs and DDTs. 2. In lake Nieuwe Meer, with the highest levels of organic micropollutants, the hepatic MFO activity was elevated in three fish species. This indicates that MFO activity might be a sensitive indicator for organic micropollutants in the aquatic environment. 3. Activities of the 3-MC type inducible isozymes were most pronounced in all three fish species studied. Though further research is required, indications for the existence of a PB-type inducible enzyme system have been demonstrated for two fish species, i.e. pike and eel. 4. Fish liver enlargement as a consequence of MFO induction could not be demonstrated in fish from the most polluted lake. 5. In fish, a correlation was observed between PCB or OCP tissue concentrations on the one hand, and hepatic MFO activities on the other. The low PAH fish/sediment ratios are further reduced when MFO systems are induced due to organic micropollution. The interrelationship between bioaccumulation and enzyme induction demonstrates the importance of an integrated study of these phenomena in field research.     

Responses of mixed-function oxygenase and antioxidase enzyme system of Mytilus sp. to organic pollution

• 1.1. Mixed-function oxidase (MFO) system components (cytochrome P-450, “418-peak”, cytochrome b₅ and NADPH-cytochrome c (P-450) reductase) and inducible antioxidant enzymes (catalase, Superoxide dismutase (SOD), glutathione peroxidase (GPX) and DT-diaphorase) has been determined in digestive glands of mussels (Mylilus galloprovincialis) collected from three Mediterranean coastal locations, exhibiting an organic pollution gradient.
• 2.2. Cytochrome P-450, the “418-peak”, catalase and SOD showed a good correlation with whole body tissue PAHs and, to a lower extent, with PCBs.
• 3.3. Microsomal NADPH-dependent DT-diaphorase, but not the NADH-dependent microsomal enzyme or the cytosolic DT-diaphorases, was indicated to increase with pollution exposure.
• 4.4. The application of such measurements to environmental monitoring is discussed. Given the magnitude of differences observed, and the state of knowledge on enzyme function and mechanisms of toxicity, a multiparameter approach is considered to offer current and future potential for detecting the impact of organic pollution on bivalve molluscs.

     

Effect of lead on the reaction of O-demethylase in p-nitro anisole in the cladoceran Moina macrocopa

The presence of the Mixed-Function Oxydase (MFO) system, in Moina macrocopa was detected through the transformation of p-nitroanisole to para-nitrophenol. The presence of this enzymatic system suggested that this cladoceran participates in the biotransformation of xenobiotics in aquatic ecosystems. This capacity, in conjunction with the aquatic flea's high tolerance to environmental stress, suggested that M. macrocopa could be used in bioremediation efforts to increase ecosystem health. The effects of lead on the MFO system in M. macrocopa, were also studied. Lead acted as an inhibitor of the enzymatic complex. Therefore, induction of MFO-activity as an early warning may not work in waterbodies affected by both inducers of MFO and inhibitors like lead.     

Altered drug metabolism in parasitic diseases

While the immune system represents the main line of host defence against parasite infections, mixed function oxidase (MFO) systems (Box 1) offer the main line of defence against drugs and other biologically active substances. But, as this review shows, many parasites can exert a profound effect on the host MFO system by altering the microsomal drug-metabolizing enzymes and electron transport carriers such as cytochrome P-450. This can markedly affect the host's ability to metabolize biologically active compounds, often with adverse physiological, pharmacological and toxicological consequences. In mammals, drug metabolism occurs predominantly in the liver, and to a lesser extent in the spleen, lungs, kidneys, intestine and cerebral tissues. Thus those parasites that occupy sites in these tissues - such as amoebae, Fasciola, schistosomes and malaria - tend to be those with greatest effects on the host's ability to metabolize drugs. The effects can modify the host response to substances unrelated to the infection, and to drugs which may be administered under a chemotherapeutic regime.     

Induction of mixed-function oxygenase system and antioxidant enzymes in the coral Montastraea faveolata on acute exposure to benzo(a)pyrene

Components of the cytochrome P(450) monooxygenase system (MFO) and antioxidant enzymes were investigated in the coral Montastraea faveolata exposed to the organic contaminant benzo(a)pyrene (B(a)P). For bioassays the corals were exposed to increasing concentrations of B(a)P (0.01 and 0.1 ppm) for 24 and 72 h, with water renewal every 24 h. Enzymatic activity of catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) were measured in host (polyp) and hosted (zooxanthellae) cells. NADPH cytochrome c reductase activity and contents of cytochrome P(450) and P(420) were only measured in the polyp. Antioxidant enzymes CAT and SOD in polyps and zooxanthellae and GST in polyps increased significantly at the highest concentration and maximum time of exposure. Cytochrome P(420) was found in all colonies, and the cytochrome P(450) content was greatest in the colonies from the highest concentrations of contaminant. NADPH cytochrome c reductase activity and the concentration of pigments did not vary between treatments. This is the first report of the induction of both detoxifying mechanisms, the MFO system and antioxidant enzymes on acute exposure to an organic contaminant in the reef-constructing coral species M. faveolata.     

From Rare to Dominant: a Fine-Tuned Soil Bacterial Bloom during Petroleum Hydrocarbon Bioremediation

Hydrocarbons are worldwide-distributed pollutants that disturb various ecosystems. The aim of this study was to characterize the short-lapse dynamics of soil microbial communities in response to hydrocarbon pollution and different bioremediation treatments. Replicate diesel-spiked soil microcosms were inoculated with either a defined bacterial consortium or a hydrocarbonoclastic bacterial enrichment and incubated for 12 weeks. The microbial community dynamics was followed weekly in microcosms using Illumina 16S rRNA gene sequencing. Both the bacterial consortium and enrichment enhanced hydrocarbon degradation in diesel-polluted soils. A pronounced and rapid bloom of a native gammaproteobacterium was observed in all diesel-polluted soils. A unique operational taxonomic unit (OTU) related to the Alkanindiges genus represented ∼0.1% of the sequences in the original community but surprisingly reached >60% after 6 weeks. Despite this Alkanindiges-related bloom, inoculated strains were maintained in the community and may explain the differences in hydrocarbon degradation. This study shows the detailed dynamics of a soil bacterial bloom in response to hydrocarbon pollution, resembling microbial blooms observed in marine environments. Rare community members presumably act as a reservoir of ecological functions in high-diversity environments, such as soils. This rare-to-dominant bacterial shift illustrates the potential role of a rare biosphere facing drastic environmental disturbances. Additionally, it supports the concept of “conditionally rare taxa,” in which rareness is a temporary state conditioned by environmental constraints.     

Insights into environmental bioremediation by microorganisms through functional genomics and proteomics

Environmental pollutants in the soil are a major concern worldwide. Bioremediation mediated by microorganisms is a highly promising technology that is environmentally friendly, safe, and effective. However, incomplete biological information regarding the cellular responses in many microbial communities restricts progress in the site-specific mineralization process. The application of proteomics in environmental bioremediation research provides a global view of the protein compositions of the microbial cells and offers a promising approach to address the molecular mechanisms of bioremediation. With the combination of proteomics, functional genomics provide an insight into global metabolic and regulatory networks that can enhance the understanding of gene functions. This article deals with the applications of functional genomics and proteomics to dissect the cellular responses to environmental stimuli, such as stress response, induction and expressions of regulatory proteins/enzymes in response to aromatic hydrocarbons and heavy metals. An understanding of the growth conditions governing the expression of the proteome (for example, enzymes and regulatory proteins of aromatic hydrocarbon degradation, energy generation pathways, transport and stress-related proteins) in a specific environment is essential for developing rational strategies for successful bioremediation.     

Hydrocarbon metabolism and cortisol balance in oil-exposed ringed seals, Phoca hispida

1. Ringed seals were exposed experimentally to oil contamination, by feeding of a [14C]naphthalene marked crude oil in fish for up to 4 days at a rate of 5 ml/day. 2. Mixed function oxygenase (MFO) activity, measured as aryl hydrocarbon hydroxylase in liver and kidney, was found to be induced, in particular in kidney tissue where the activity increased 3-fold. 3. MFO induction correlated with a high degree of conversion of crude oil hydrocarbons to water-soluble metabolites. Most of the radioactivity was found in the polar fraction of plasma and urine. 4. Plasma cortisol levels were somewhat elevated by captive holding, and increased markedly after oil-exposure. Cortisol half-life decreased after oil exposure from 1 3/4 to 1 hr.     

Application of a Bayesian nonparametric model to derive toxicity estimates based on the response of Antarctic microbial communities to fuel‐contaminated soil

Ecotoxicology is primarily concerned with predicting the effects of toxic substances on the biological components of the ecosystem. In remote, high latitude environments such as Antarctica, where field work is logistically difficult and expensive, and where access to adequate numbers of soil invertebrates is limited and response times of biota are slow, appropriate modeling tools using microbial community responses can be valuable as an alternative to traditional single‐species toxicity tests. In this study, we apply a Bayesian nonparametric model to a soil microbial data set acquired across a hydrocarbon contamination gradient at the site of a fuel spill in Antarctica. We model community change in terms of OTUs (operational taxonomic units) in response to a range of total petroleum hydrocarbon (TPH) concentrations. The Shannon diversity of the microbial community, clustering of OTUs into groups with similar behavior with respect to TPH, and effective concentration values at level x, which represent the TPH concentration that causes x% change in the community, are presented. This model is broadly applicable to other complex data sets with similar data structure and inferential requirements on the response of communities to environmental parameters and stressors.     

Differential sensitivity of CYP1A to 3,3′,4′,4-tetrachlorobiphenyl and benzo(a)pyrene in two Lepomis species

Although Lepomis species are abundant in a wide variety of habitats throughout North America and could serve as potentially valuable biomonitoring tools, few studies have examined the induction of pollutant biomarkers in this genus. We hypothesized that the induction of cytochrome P-450 1A (CYP1A), a sensitive and widely used indicator of response to aquatic contaminants, would serve as an effective biomarker of organic pollutant exposure in Lepomis species. We examined the response of CYP1A and two of the major pollutant-responsive phase II enzymes, glutathione S-transferase (GST), and uridine diphosphate glucuronyltransferase (UDPGT), in Lepomis exposed to organic pollutants under laboratory and field conditions. Two Lepomis species (longear sunfish, Lepomis megalottis and bluegill, Lepomis macrochirus) were exposed in the laboratory via intraperitoneal injection to corn oil (vehicle), benzo(a)pyrene (BaP) (10 and 50 mg/kg), a polynuclear aromatic hydrocarbon (PAH) or 3,4,3′,4′-tetrachlorobiphenyl (PCB 77) (0.1 and 1.0 mg/kg), a dioxin-like planar halogenated aromatic hydrocarbon (HAH), and sacrificed 2 (BaP) or 7 (corn oil, PCB77) days later. Lepomis hepatic CYP1A exhibited differential sensitivity to these two classes of environmental contaminants. CYP1A activity was weakly induced in bluegill exposed to 1.0 mg/kg PCB 77 (3 fold induction over controls) but strongly induced in both bluegill and longear sunfish exposed to 50 mg/kg BaP (37 and 15 fold induction over controls, respectively). In contrast, hepatic GST activity in both species remained unchanged following the treatment with either compound and hepatic UDPGT activity, which was assessed only in BaP-treated longear sunfish, was unaffected by that chemical, indicating these phase II enzymes may not be sensitive pollutant biomarkers in this genus. Further, longear sunfish collected from a PCB contaminated site displayed relatively low levels of CYP1A activity despite PCB body burdens associated with strong induction of CYP1A activity in other fish species. The strong induction of CYP1A by BaP with much weaker CYP1A response to PCB indicates that CYP1A in Lepomis sp. could be an excellent biomarker for PAH pollution, but may not be a reliable indicator of site contamination by halogenated hydrocarbons. We conclude that Lepomis species provide a useful model for examining the regulation and potential consequences of differential pollutant sensitivity, but that CYP1A in these species should be used with caution as an indicator of halogenated contaminants.     

Towards a specific index of impact by organic pollution for marine invertebrates

• 1.1. The responses of the mixed-function oxygenase (MFO) system in marine invertebrates to organic xenobiotics are limited, although some field and mesocosm successes have been recorded. Activity of 7-ethoxyresorufin O-deethylase (EROD) is low or undetectable.
• 2.2. Antioxidant enzymes and other biochemical parameters, although less specific than the MFO system, may serve as complementary or supportive measurements in environmental impact assessment.
• 3.3. By analogy with cytochrome P-450 IA1 of fish, confident application of biochemical systems in environmental monitoring will depend upon their characterization and an understanding of the regulatory processes involved. A single “EROD-type” measurement may or may not be possible, but several complementary ones may suffice.

     

Evolutionally guided enzyme design

A combination of "rational" and "irrational" strategies for the creation of enzymes with novel properties is proving to be a powerful concept in the field of enzyme engineering. Guided by principles of physical organic chemistry, rational design strategies are used to identify suitable target enzymes and to choose appropriate molecular biological methods for engineering purposes. In contrast, irrational (or random) strategies are centered around the biological paradigm of stochastic molecular evolution. As illustrated in this review, such a hybrid approach is particularly useful for the design of new modular enzymes. (c) 1996 John Wiley & Sons, Inc.     

Comparative studies of lipase and phospholipase A2 acting on substrate monolayers.

The kinetic aspects of lipolysis by pancreatic lipase and phospholipase A2 from different sources have been compared using monomolecular films of short chain lipids as the substrates. Phosphatidylcholine monolayers, in contrast to phosphatidylethanolamine and phosphatidylglycerol monolayers, were resistant to hydrolysis by pancreatic lipase. The induction time, measured during pre-steady state conditions, increased abruptly for a given value of the surface pressure. This appears to be due to a degree of lipid packing above which the enzyme no longer can penetrate the lipid film. The existence of an optimum in the velocity versus surface pressure profile is the result of at least two counterbalancing factors. As the surface pressure increases, the amount of enzyme present in the interface decreases, whereas the minimal specific activity of the enzyme increases. From this study with monolayers we can conclude that activity of lipolytic enzymes used as tools for probing biological membranes will be greatly influenced by the physiochemical nature of the membrane-water interface. Thus, studies such as this one which can measure the penetrating ability of various lipolytic enzymes can be useful in deriving a better understanding of biological membrane structure.