Post-synthetic modification of proteins in pathology (defective proteins)

Process of proteins' modification under pathology are considered and classified. Attention is focused on the processes of postsynthetic modification. It is suggested not only proteins with the structure different from that in the norm to be considered as defective but also those whose biosynthesis does not provide the fulfilment of a definite function by them. Processes of proteins damaging by certain metabolites and xenobiotics are considered in detail. It is shown that all proteins of the organism may be damaged both by the excess of the natural metabolite (glucose) and by xenobiotics. The hypothesis is advanced on the important role of albumin as a primary barrier in the organism response on the chemical affection. The cooperative mechanism of the ligands' binding which underlies the detoxication function of the albumin is postulated.     

Enzymatic System of Metabolism and Detoxication of Xenobiotics as a Basis of Metabolic Portraiting during Prognostication of Risk of Pathological Processes

We studied intraspecific features of the main enzymes of metabolism and detoxication of xenobiotics on mice (eight inbred lines) and rats (five lines) for estimation of possible variants of complete or incomplete metabolic equality. Significant genetically determined intraspecific differences for activities of the enzymes of metabolism and detoxication of xenobiotics were described. Generalized criteria for comparison of the metabolic status were proposed on the basis of activities of the main enzymes: cytochrome P-450 (hydroxylation and epoxidation), epoxyhydrolase, glutathione-S-transferase, UDP-glucuronosyl transferase, and sulfotransferase. The proposed criteria for estimation of the metabolic parameters of an individual can serve as a basis of metabolic portraiting.     

In situ sites for xenobiotic activation and detoxication: implications for the differential susceptibility of cells to the toxic actions of environmental chemicals

The findings reported in this communication illustrate that histochemical approaches can provide a significant amount of insight into an area of considerable toxicologic importance. Results of our immunohistochemical and histochemical studies clearly demonstrate that neither xenobiotic-metabolizing enzymes nor oxidative xenobiotic metabolism occur uniformly throughout tissues that often are damaged as a result of the bioactivation of environmental chemicals and other xenobiotics, that there can be significant differences in both the contents and activities of xenobiotic-metabolizing enzymes among even morphologically similar cells such as hepatocytes, and that enzyme inducers can alter differentially the extents to which different cells in a tissue metabolize xenobiotics. Knowledge of the precise intratissue localizations and distributions of xenobiotic-metabolizing enzymes and xenobiotic biotransformation reactions clearly is critical for defining the roles individual cells play in the metabolism of xenobiotics. It must be recognized, however, that the mere presence of xenobiotic-metabolizing enzymes in a cell cannot, by itself, explain why that cell might be highly susceptible to toxicities resulting from the bioactivation of certain xenobiotics. Thus, it is apparent that considerably more study is needed, especially in situ using histologic and cytologic techniques, in order to characterize the balance between xenobiotic activation and detoxication processes within individual cells in target tissues and elucidate the basis for the cell-selective nature of toxicities caused by the generation of reactive metabolites from many xenobiotics.     

The relevance of xenobiotic metabolism in the interindividual susceptibility to chemicals

Biotransformation enzymes may catalyze either detoxication or bioactivation reactions; indeed, many xenobiotics exert their toxic effects after metabolic activation to electrophilic chemicals, interacting with nucleophilic sites on cellular macromolecules. On the other hand, by increasing xenobiotic hydrophilicity, the drug-metabolizing enzymes favors excretion of lipophilic chemicals, not allowing their bioaccumulation up to toxic levels. The expression of the enzymes of the drug-metabolizing system is modulated by genetic, pathological, developmental, environmental and dietary factors. Genetic polymorphism resulting in interindividual and interethnic variation in xenobiotic metabolism is responsible for differences in the susceptibility to chemical-induced toxicity and carcinogenicity, allowing the identification of people at increased risk. Moreover, differences in drug metabolism may correspond to variability in drug response during pharmacological therapy, which can be manifest either as adverse reactions or as a lack of benefit.     

Drug metabolizing enzymes in cerebrovascular endothelial cells afford a metabolic protection to the brain.

The brain is partially protected from chemical insults by a physical barrier mainly formed by the cerebral microvasculature, which prevents penetration of hydrophilic molecules in the cerebral extracellular space. This results from the presence of tight junctions joining endothelial cells, and from a low transcytotic activity in endothelial cells, inducing selective permeability properties of cerebral microvessels that characterize the blood-brain barrier. The endothelial cells provide also, as a result of their drug-metabolizing enzymes activities, a metabolic barrier against potentially penetrating lipophilic substances. It has been established that in cerebrovascular endothelial cells, several families of enzymes metabolize potentially toxic lipophilic substrates from both endogenous and exogenous origin to polar metabolites, which may not be able to penetrate further across the blood-brain barrier. Enzymes of drug metabolism present at brain interfaces devoid of blood-brain barrier, like circumventricular organs, pineal gland, and hypophysis, that are potential sites of entry for xenobiotics, display higher activities than in cerebrovascular endothelial cells, and conjugation activities are very high in the choroid plexus. Finally, xenobiotic metabolism normally results in detoxication, but also in some cases in the formation of pharmacologically active or neurotoxic products, possibly altering some blood-brain barrier properties.     

Activity of phenolsulfotransferases in the human gastrointestinal tract

Sulfate conjugation by sulfotransferase enzymes is an important pathway for the detoxication of xenobiotics and endogenous compounds. The large surface area of the gastrointestinal tract exposes the body to a range of potential toxins, and hence local metabolism is likely to be important. The ability of different regions of the gut to sulfate micromolar concentrations of simple phenols and catecholamines has been determined throughout the gut using 4-nitrophenol and dopamine as standard substrates. The pattern of sulfation of both compounds was similar, with activity highest in the small bowel >right colon >left colon >rectum >stomach >esophagus. High concentrations of sulfotransferases in the reservoir areas of the right and left colon indicate possible importance in detoxication by sulfation and also perhaps in activating mutagens in the same areas. Nutritional factors, such as a high-fat diet may, however, alter sulfotransferase activity.     

Dietary effects on cytochromes P450, xenobiotic metabolism, and toxicity.

The levels and activities of cytochrome P450 enzymes are influenced by a variety of factors, including the diet. In this article, the effects of selected non-nutritive dietary chemicals, macronutrients, micronutrients, and ethanol on cytochromes P450 and xenobiotic metabolism are reviewed in the light of our current understanding of the multiplicity and substrate specificity of cytochrome P450 enzymes. Although the mechanisms of action of several dietary chemicals on specific cytochrome P450 isozymes have been established, those for macro- and micronutrients are largely unknown. It is known, however, that specific nutrients may have varied effects on different cytochrome P450 forms and thus may affect the metabolism of various drugs differently. Nutritional deficiencies generally cause lowered rates of xenobiotic metabolism. In certain cases, such as thiamin deficiency and mild riboflavin deficiency, however, enhanced rates of metabolism of xenobiotics were observed. The effects of dietary modulation of xenobiotic metabolism on chemical toxicity and carcinogenicity are discussed.     

Inevitable glutathione,then and now

Glutathione a predominant tripeptide thiol compound of many prokaryotes and eukaryotes, is synthesized from its precursor amino acids eg. gamma-glutamate, cysteine and glycine. It is mainly involved in detoxication mechanisms through conjugation reactions. Other functions include thiol transfer, destruction of free radicals and metabolism of various exogenous and endogenous compounds. It becomes mandatory for a cell to manage high concentration of intracellular GSH to protect itself from chemical/dug abuse. Glutathione dependent enzymes viz: glutathione-S-transferases, glutathione peroxidase, glutathione reductase and gamma-glutamate transpeptidase facilitate protective manifestations. Liver serves as a glutathione-generating factor which supplies the kidney and intestine with other constituents of glutathione resynthesis. The principal mechanism of hepatocyte glutathione turnover appears to be cellular efflux. Kidney too plays an important role in organismic GSH homeostasis. Role of GSH in organs like lung, intestine and brain has recently been described. GSH involvement in programmed cell death has also been indicated. Immense interest makes the then "thee glutathione" as "inevitable glutathione". This article describes the role of this vital molecule in cell physiology and detoxication mechanisms in particular.     

Forging the links between metabolism and carcinogenesis

Metabolism plays important roles in chemical carcinogenesis, both good and bad. The process of carcinogen metabolism was first recognized in the first half of the twentieth century and developed extensively in the latter half. The activation of chemicals to reactive electrophiles that become covalently bound to DNA and protein was demonstrated by Miller and Miller [Cancer 47 (1981) 2327]. Today many of the DNA adducts formed by chemical carcinogens are known, and extensive information is available about pathways leading to the electrophilic intermediates. Some concepts about the stability and reactivity of electrophiles derived from carcinogens have changed over the years. Early work in the field demonstrated the ability of chemicals to modulate the metabolism of carcinogens, a phenomenon now described as enzyme induction. The cytochrome P450 enzymes play a prominent role in the metabolism of carcinogens, both in bioactivation and detoxication. The conjugating enzymes can also play both beneficial and detrimental roles. As an example of a case in which several enzymes affect the metabolism and carcinogenicity of a chemical, aflatoxin B1 (AFB1) research has revealed insight into the myriad of reaction chemistry that can occur even with a 1s half-life for a reactive electrophile. Further areas of investigation involve the consequences of enzyme variability in humans and include areas such as genomics, epidemiology, and chemoprevention.     

人羧酯酶的研究进展

羧酯酶是一类可与有机磷化合物结合且活性受抑制的B－酯酶,分布很广,能水解许多羧酯类、酰胺类、硫酯类物质,其天然底物尚未清楚,故其生理功能仍在研究中,可能与脂质代谢,药物或毒物的生物转化有关．对羧酯酶的一级结构及基因序列的研究表明,羧酯酶是由许多生化特性不同的同工酶组成．     

Mixed function oxygenases and xenobiotic detoxication/toxication systems in bivalve molluscs

Components of a xenobiotic detoxication/toxication system involving mixed function oxygenases are present inMytilus edulis. Our paper critically reviews the recent literature on this topic which reported the apparent absence of such a system in bivalve molluscs and attempts to reconcile this viewpoint with our own findings on NADPH neotetrazolium reductase, glucose-6-phosphate dehydrogenase, aldrin epoxidation and other reports of the presence of mixed function oxygenases. New experimental data are presented which indicate that some elements of the detoxication/toxication system inM. edulis can be induced by aromatic hydrocarbons derived from crude oil. This includes a brief review of the results of long-term experiments in which mussels were exposed to low concentrations of the water accommodated fraction of North Sea crude oil (7.7–68 μg 1⁻¹) in which general stress responses such as reduced physiological scope for growth, cytotoxic damage to lysosomal integrity and cellular damage are considered as characteristics of the general stress syndrome induced by the toxic action of the xenobiotics. In addition, induction in the blood cells of microsomal NADPH neotetrazolium reductase (associated with mixed function oxygenases) and the NADPH generating enzyme glucose-6-phosphate dehydrogenase are considered to be specific biological responses to the presence of aromatic hydrocarbons. The consequences of this detoxication/toxication system forMytilus edulis are discussed in terms of the formation of toxic electrophilic intermediate metabolites which are highly reactive and can combine with DNA, RNA and proteins with subsequent damage to these cellular constituents. Implications for neoplasms associated with the blood cells are also discussed. Finally, in view of the increased use of mussel species in pollutant monitoring programmes, the induction phenomenon which is associated with microsomal enzymes in the blood cells is considered as a possible tool for the detection of the biological effects of environmental contamination by low concentrations of certain groups of organic xenobiotics.     

Metabolic disturbances in fish exposed to sodium pentachlorophenate (NaPCP) and 3,3',4,4',5-pentachlorobiphenyl (PCB126), individually or combined

The Swan River Estuary is the recipient of multiple urban and agricultural contaminants which have the potential to induce liver detoxication enzymes as well as altering the metabolism of aquatic organisms. To test if altered liver metabolism would influence liver detoxication capacities, pink snapper (Pagrus auratus) were i.p. injected with peanut oil (controls), or pentachlorobiphenyl #126 (PCB126), with sodium pentachlorophenate (NaPCP), or PCB126+NaPCP. Relative to controls, ethoxyresorufin-O-deethylase (EROD) activity was induced in the PCB126 and PCB126+NaPCP fish, but not in the NaPCP group. In the liver, cytochrome c oxidase (CCO) activity was enhanced by the treatments while citrate synthase (CS) activity remained unchanged and lactate dehydrogenase (LDH) activity was increased in the NaPCP treatment only. The results suggest that liver CCO activity may be a suitable biomarker of effect following exposure to PCBs or phenolic compounds. In the white muscle, only the PCB126+NaPCP treatment enhanced CCO activity, with all other enzymatic activities remaining unchanged. It appears that the resilience to metabolic perturbations is greater for white muscle than for liver. Low serum sorbitol dehydrogenase (sSDH) activity and histopathology of the liver indicated no significant alteration of cellular structure, albeit the lipid droplet size was increased in the PCB126 and in the PCB126+NaPCP treatments. It is concluded that the hepatic metabolic changes correspond to histopathological observations, but an altered metabolic capacity do not influence the metabolism of xenobiotics by liver enzymes, as measured by EROD activity.     

Enzymatic reactions in biotechnology (48th Bach lecture)]

The paper is the 48th Bach Lecture presented under the same title. It covers the biochemical mechanisms of the biogenesis of microbial biosynthetic products, role of acetyl-CoA, function of the succinate-glycine cycle, reactions of the hexose-monophosphate pathway of carbon metabolism. The reversible action of hydrolases in enzymatic catalysis and degradation of xenobiotics are discussed. The data on redox reactions are pooled. Such modern biotechnological processes as epoxidation, synthesis of acrylamide and some monomers involved in chemical syntheses of polymers, synthesis of oligosaccharide and fluorine-containing amino acids are considered. Promising commercial applications of biocatalysis are discussed.     

Xenobiotic metabolizing enzymes in the central nervous system: Contribution of cytochrome P450 enzymes in normal and pathological human brain

The metabolism of xenobiotics in human brain constitutes a field of recent intensive research in relation to the potential implications in the pharmacological effect of drugs acting on the central nervous system. Cytochrome P450 enzymes (CYPs) play a crucial role in these metabolic pathways and the existence of functional CYP monooxygenases in brain is now well established. These enzymes are preferentially localized in the neuronal cells within the microsomal fraction and the inner membrane of mitochondria. Although low, the metabolism in situ could influence individual response to xenobiotics or produce reactive, toxic metabolites causing irreversible damage in the neuronal cells. The abundant presence of CYPs in selective cell populations within different regions of the brain has also suggested a role for these enzymes in brain physiology thus not restricted to xenobiotic-induced neurotoxicity. For instance, CYPs participate in the regulation of neurotransmitters and steroids and brain maintenance of cholesterol homeostasis. Recent advances support an additional role for these enzymes in the pathogenesis of psychiatric and neurodegenerative disorders such as depression, schizophrenia, and Alzheimer's and Parkinson's diseases. The characterization of brain CYP isoforms and their localization, the identification of their substrates and metabolic end-products will allow better understanding of the role of these enzymes in brain physiology, development and diseases.     

Effect of various supplies of vitamin B2 in the rat body on the activity of enzymes participating in the metabolism of xenobiotics

Studies on the effect of different content of vitamin B2 (alimentary deficiency, additional administration) in the rat organism on the activity of enzymes participating in the metabolism of foreign substances and on the inducing effect of phenobarbital have shown that vitamin B2 to a considerable extent controls the activity of flavin-containing enzymes participating in the metabolism of xenobiotics (D-amino acid oxidase, xanthine and aldehyde oxidases, NADH- and NADPH-reductase activity of neotetrazolium) and a number of enzymes for which flavins do not play the role of prosthetic group (esterases aldehyde and formaldehyde dehydrogenases, demethylase and hydroxylase). Different content of vitamin B2 in animal organism also influences the action of phenobarbital, an inductor of xenobiotics metabolism, and the acetanilide biotransformation rate.     

Enzymatic Detoxication,Conformational Selection,and the Role of Molten Globule Active Sites

The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes.     

Metabolism and toxicity of xenobiotics in the adrenal cortex, with particular reference to 7,12-dimethylbenz(a)anthracene

The adrenal cortex contains high amounts of detoxifying enzymes, as well as generators and protectors of reactive oxygen species. The high content of cytochrome P-450 enzymes in the adrenal cortex together with its remarkable tendency to accumulate hydrophobic substances probably contributes to the extraordinary vulnerability of the gland to a number of xenobiotics. The best studied adrenocorticolytic compounds are the potent carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) and its liver metabolite 7-hydroxymethyl-12-methylbenz(a)anthracene (7-OHM-12-MBA). Adrenocorticolysis generated by these agents in vivo as well as in vitro demonstrates high regioselective requirements and is strongly influenced by the presence of ACTH, steroids, cytochrome P-450 inhibitors and antioxidants. Furthermore, 7-OHM-12-MBA has been demonstrated to uniquely generate selective and massive oxidation of mitochondrial glutathione in cultured rat adrenal cells. The DMBA-induced adrenocorticolysis is thoroughly discussed in this review with particular emphasis on the metabolism of DMBA and the influence of various effectors. A working hypothesis involving a possible peroxidative mechanism is also presented.     

Metabolism and toxicity of xenobiotics in the adrenal cortex,with particular reference to 7,12-dimethylbenz(a) anthracene

The adrenal cortex contains high amounts of detoxifying enzymes, as well as generators and protectors of reactive oxygen species. The high content of cytochrome P-450 enzymes in the adrenal cortex together with its remarkable tendency to accumulate hydrophobic substances probably contributes to the extraordinary vulnerability of the gland to a number of xenobiotics. The best studied adreno-corticolytic compounds are the potent carcinogen 7, 12-dimethylbenz(a)anthracene (DMBA) and its liver metabolite 7-hydroxymethyl-12-methylbenz (a)anthracene (7-OHM-12-MBA). Adrenocorticolysis generated by these agents in vivo as well as in vitro demonstrates high regioselective requirements and is strongly influenced by the presence of ACTH, steroids, cytochrome P-450 inhibitors and antioxi-dants. Furthermore, 7-OHM-12-MBA has been demonstrated to uniquely generate selective and massive oxidation of mitochondrial glutathione in cultured rat adrenal cells. The DMBA-induced adrenocorticolysis is thoroughly discussed in this review with particular emphasis on the metabolism of DMBA and the influence of various effectors. A working hypothesis involving a possible peroxidative mechanism is also presented.     

Characterisation of two novel CYP4 genes from the marine polychaete Nereis virens and their involvement in pyrene hydroxylase activity

Cytochrome P450 enzymes (CYP enzymes) catalyse the initial step in biotransformation of xenobiotics like polycyclic aromatic hydrocarbons (PAHs). The marine polychaete Nereis virens has a high capacity for biotransformation of PAHs. In the present study, the complete cDNA sequences of two novel CYP genes isolated from N. virens gut tissue are reported. One named CYP342A1, the first member of a new family and the other named CYP4BB1, the first member of a new subfamily. This is the first investigation of specific CYP enzymes from marine polychaetes in which catalytic activity has been determined. Both CYP enzymes had monooxygenase activity and catalysed hydroxylation of pyrene to 1-hydroxypyrene. Based on the present results it is likely that both CYP4BB1 and CYP342A1 are involved in xenobiotic biotransformation. Furthermore, site-directed mutagenesis of the conserved cysteine residue of the heme binding domain resulted in complete loss of monooxygenase activity of both CYP enzymes, indicating that this cysteine residue is indispensable for monooxygenase activity of invertebrate CYP enzymes, as has been well documented in vertebrates. Considering the important role of CYP enzymes in biotransformation of xenobiotics and the presence of N. virens in estuarine environments that accumulates organic xenobiotics, our results are important in understanding the molecular mechanism of biotransformation in marine polychaetes.