Biomedical subjects

M E Herrero

Publications and source records attributed to M E Herrero.

6 recordsLinked to original sources

PubMed2001

Sequestration of biological reactive intermediates by trapping as covalent enzyme-intermediate complex.

One important class of biological reactive intermediates arising in the course of human xenobiotic metabolism are arene and alkene oxides. The major safeguard against the potential genotoxic effects of these compounds is the microsomal epoxide hydrolase (mEH). This enzyme has a broad substrate specificity but--on the first sight--seems to be inadequately suited for this protection task due to its low turnover number with most of its substrates. The recent progress in the understanding of the mechanism of enzymatic epoxide hydrolysis has shed new light on this apparent dilemma: Epoxide hydrolases convert their substrates via the intermediate formation of a covalent enzyme-substrate complex, and it has been shown that the formation of the intermediate proceeds by orders of magnitudes faster than the subsequent hydrolysis, i.e. the formation of the terminal product. Thus, the enzyme acts like a molecular sponge by binding and inactivating the dangerous metabolite very fast while the subsequent product release is considerably slower, and quantification of the latter heavily underestimates the speed of detoxification. Usually, the slow enzyme regeneration does not pose a problem, since the mEH is highly abundant in human liver, the organ with the highest capacity to metabolically generate epoxides. Computer simulation provides evidence that the high amount of mEH enzyme is crucial for the control of the steady-state level of a substrate epoxide and can keep it extremely low. Once the mEH is titrated out under conditions of extraordinarily high epoxide concentration, the epoxide steady-state level steeply rises, leading to a sudden burst of the genotoxic effect. This prediction of the computer simulation is in perfect agreement with our experimental work. V79 Chinese Hamster cells that we have genetically engineered to express human mEH at about the same level as that observed in human liver are well protected from any measurable genotoxic effect of the model compound styrene oxide (STO) up to an apparent threshold level of 100 microM in the cell culture medium. In V79 cells that do not express mEH, STO triggers the formation of DNA strand breaks in a dose-dependent manner with no apparent threshold. Above 100 microM, the genotoxic effect of STO in the mEH-expressing cell line parallels the one in the parental cell line.

Animals↗

PubMed1998

Polymorphisms of N-acetyltransferases, glutathione S-transferases, microsomal epoxide hydrolase and sulfotransferases: influence on cancer susceptibility.

It has become clear that several polymorphisms of human drug-metabolizing enzymes influence an individual's susceptibility for chemical carcinogenesis. This review gives an overview on relevant polymorphisms of four families of drug-metabolizing enzymes. Rapid acetylators (with respect to N-acetyltransferase NAT2) were shown to have an increased risk of colon cancer, but a decreased risk of bladder cancer. In addition an association between a NAT1 variant allele (NAT*10, due to mutations in the polyadenylation site causing approximately two fold higher activity) and colorectal cancer among NAT2 rapid acetylators was observed, suggesting a possible interaction between NAT1 and NAT2. Glutathione S-transferases M1 and T1 (GSTM1 and GSTT1) are polymorphic due to large deletions in the structural gene. Meta-analysis of 12 case-control studies demonstrated a significant association between the homozygous deletion of GSTM1 (GSTM1-0) and lung cancer (odds ratio: 1.41; 95% CI: 1.23-1.61). Combination of GSTM1-0 with two allelic variants of cytochrome P4501A1 (CYP1A1), CYP1A1 m2/m2 and CYP1A1 Val/Val further increases the risk for lung cancer. Indirect mechanisms by which deletion of GSTM1 increases risk for lung cancer may include GSTM1-0 associated decreased expression of GST M3 and increased activity of CYP1A1 and 1A2. Combination of GST M1-0 and NAT2 slow acetylation was associated with markedly increased risk for lung cancer (odds ratio: 7.8; 95% CI: 1.4-78.7). In addition GSTM1-0 is clearly associated with bladder cancer and possibly also with colorectal, hepatocellular, gastric, esophageal (interaction with CYP1A1), head and neck as well as cutaneous cancer. In individuals with the GSTT1-0 genotype more chromosomal aberrations and sister chromatid exchanges (SCEs) were observed after exposure to 1,3-butadiene or various haloalkanes or haloalkenes. Evidence for an association between GSTT1-0 and myelodysplastic syndrome and acute lymphoblastic leukemia has been presented. A polymorphic site of GSTP1 (valine to isoleucine at codon 104) decreases activity to several carcinogenic diol epoxides and was associated with testicular, bladder and lung cancer. Microsomal expoxide hydrolase (mEH) is polymorphic due to amino acid variation at residues 113 and 139. Polymorphic variants of mEH were associated with hepatocellular cancer (His-113 allele), ovarian cancer (Tyr-113 allele) and chronic obstructive pulmonary disease (His-113 allele). Three human sulfotransferases (STs) are regulated by genetic polymorphisms (hDHEAST, hM-PST, TS PST). Since a large number of environmental mutagens are activated by STs an association with human cancer risk might be expected.

Arylamine N-Acetyltransferase↗

PubMed1997

Recombinant expression of human microsomal epoxide hydrolase protects V79 Chinese hamster cells from styrene oxide- but not from ethylene oxide-induced DNA strand breaks.

Styrene 7,8-oxide and ethylene oxide are widely used genotoxic bulk chemicals, which have been associated with potential carcinogenic hazard for occupationally exposed workers. Both epoxides alkylate DNA preferentially at the N-7 position of guanine and consequently produce single-strand breaks and alkali labile sites in the DNA of exposed cells. In order to study the role of human microsomal epoxide hydrolase (hmEH) in protecting cells against genotoxicity of styrene 7,8-oxide and ethylene oxide, we expressed the cDNA of hmEH in V79 Chinese hamster cells. We obtained a number of cell clones that expressed functionally active epoxide hydrolase. Among these, the clone 92hmEH-V79 revealed an especially high enzymatic mEH activity toward styrene 7,8-oxide (10 nmol converted per mg of protein per min, measured in the 9,000 x g supernatant of the cell homogenate), that was 100 times higher than that determined in mock-transfected cells and within the range of mEH activity in human liver. Styrene 7,8-oxide-induced DNA single-strand breaks/alkali labile sites (dose range 10 microM to 1 mM styrene 7,8-oxide) measured by the alkaline elution technique were significantly lower in the 92hmEH-V79 cells as compared to the mock-transfected cells. The protection against styrene 7,8-oxide genotoxicity in 92hmEH-V79 cells could be abolished by addition of valpromide, a selective inhibitor of microsomal epoxide hydrolase. These results clearly show that the metabolism of styrene 7,8-oxide by hmEH in 92hmEH-V79 cells was responsible for the protection against styrene 7,8-oxide genotoxicity. On the other hand, no protective effect of epoxide hydrolase expression could be observed on ethylene oxide-induced DNA damage with the recombinant cell line over a dose range of 0.5-2.5 mM ethylene oxide. This selectivity of the protective effect on epoxide genotoxicity thus appears to be an important factor that must be taken into account for the prediction of the genotoxic risk of epoxides themselves or compounds that can be metabolically activated to epoxides.

Animals↗

PubMed1995

Fluorometric microassay to quantify microsomal epoxide hydrolase in 96-well plates.

The microfluorometric assay, suitable for measuring microsomal epoxide hydrolase activity in cultured cells, is based on the conversion of benzo[a]pyrene-7,8-epoxide to the corresponding trans-benzo[a]pyrene-7,8-dihydrodiol, a compound that fluoresces at 403 nm when excited at 365 nm. Activity is determined by incubating S9 fractions obtained from microcultures with this epoxide and monitoring the fluorescence with a microplate reader. Under the assay conditions selected, the photodecomposition of the reaction product was minimized and the linearity of the reaction was extended. The major advantages of this method are: (1) high sensitivity with a detection limit of 5 pmol/well of trans-benzo[a]pyrene-7,8-dihydrodiol formed, which is comparable to the most sensitive radioactive methods; (2) minimal sample requirement (1-5 micrograms liver microsomes; 10-50 micrograms S9 fraction from cultured cells); (3) reduced consumption of hazardous reagents; and (4) a considerable reduction in assay time and facility for simultaneous determination of enzyme activity in multiple samples.

Animals↗

PubMed1990

Specific treatment demand as a definitory trait of a typology in heroin addicts: differential profile of two subpopulations.

Several sociodemographic, drug use pattern, and clinical variables were investigated in two groups of heroin addicts. Inclusion in one group or the other depended on the addict's attitude toward or against receiving treatment for the addiction. Those who rejected treatment differed significantly in the following: they were younger and belonged to larger families; they started cannabis and amphetamine use earlier, and used for a longer time; they used cocaine and heroin more frequently, and alcohol less frequently; and they suffered more often from a number of somatic and personality disorders. Implications for future research on the addiction are discussed.

Adolescent↗

PubMedDate not supplied

Metabolic detoxification: implications for thresholds.

The fact that chemical carcinogenesis involves single, isolated, essentially irreversible molecular events as discrete steps, several of which must occur in a row to finally culminate in the development of a malignancy, rather suggests that an absolute threshold for chemical carcinogens may not exist. However, practical thresholds may exist due to saturable pathways involved in the metabolic processing, especially in the metabolic inactivation, of such compounds. An important example for such a pathway is the enzymatic hydrolysis of epoxides via epoxide hydrolases, a group of enzymes for which the catalytic mechanism has recently been established. These enzymes convert their substrates via the intermediate formation of a covalent enzyme-substrate complex. Interestingly, the formation of the intermediate proceeds faster by orders of magnitude than the subsequent hydrolysis, ie, the formation of the terminal product. Under normal circumstances, this does not pose a problem, since the microsomal epoxide hydrolase (mEH), the epoxide hydrolases with the best documented importance in the metabolism of carcinogens, is highly abundant in the liver, the organ with the highest capacity to metabolically generate epoxides. Computer simulation provides evidence that the high amount of mEH enzyme is favorable for the control of the steady-state level of a substrate epoxide and can keep it extremely low. However, once the mEH is titrated out under conditions of extraordinarily high epoxide concentration, the epoxide steady-state level steeply rises, leading to a sudden burst of the genotoxic effect of the noxious agent. This prediction of the computer simulation is nicely supported by experimental work. V79 Chinese hamster cells that we have genetically engineered to express human mEH at about the same level as that observed in human liver are completely protected from any measurable genotoxic effect of the model compound styrene oxide (STO) up to a dose of 100 microM in the cell culture medium (toxicokinetic threshold). In V79 cells that do not express mEH, STO leads to the formation of DNA strand breaks in a dose-dependent manner with no toxicokinetic threshold observable. Above 100 microM, the genotoxic effect of STO in the mEH-expressing cell line parallels the one in the parental cell line. Thus, the saturable protection from STO-induced strand breaks by mEH represents a typical example of a practical threshold. However, it must be pointed out that even in the presence of protective amounts of mEH, a minute but definite level of STO is present that does not contribute sufficiently to the strand break formation to overcome the background noise of the detection procedure. As pointed out above, absolute thresholds probably do not exist in chemical carcinogenesis.

Animals↗