Biomedical subjects

M Manno

Publications and source records attributed to M Manno.

At least 37 records · Page 2Linked to original sources

PubMed2001

Inactivation of rat liver cytochrome P450 (P450) by N,N-dimethylformamide and N,N-dimethylacetamide.

N,N-dimethylformamide (DMF), an organic solvent widely used in industry, is bioactivated by cytochrome P450 (P450) to reactive metabolites which are believed to be responsible for the hepatotoxicity observed in animals and humans. A decrease of the activating enzyme has been reported in rats treated with DMF, although the specific P450 isoform(s) involved and the nature of the reactive species responsible for this and the other toxic effects are still being investigated. In the present work, the effect of DMF and of the structurally related N,N-dimethylacetamide (DMAc) on the activating enzyme and the nature of the reactive species involved in the mechanism of P450 inactivation by the two chemicals were investigated in vitro. Incubation of liver microsomes from pyridine-induced rats with either substrate resulted in a dose-dependent (0-20 mM) loss of P450 (up to 28 and 24% for DMF and DMAc, respectively), microsomal haem (up to 24 and 20% for DMF and DMAc, respectively), but not protoporphyrin IX content. Moreover, bubbling of CO through the incubation mixture gave almost complete protection against substrate-dependent P450 inactivation, and the spin trapping agent N-tert-butyl-alpha-phenylnitrone, but neither glutathione nor vitamin C, provided a significant protection against DMF- or DMAc-dependent haem loss. Finally, electron spin resonance analysis of microsomal incubations in presence of DMF or DMAc showed spectral evidence for a carbon centered radical intermediate. The results indicate, overall, that both compounds are metabolized in vitro by P450, probably CYP2E1, to free radical metabolites which attack the haem prosthetic group, leading to suicidal enzyme inactivation.

Acetamides↗

PubMed2001

Bioactivation and toxicity in vitro of HCFC-123 and HCFC-141b: role of cytochrome P450.

The bioactivation and cytotoxicity in vitro of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) and 1,1-dichloro-1-fluoroethane (HCFC-141b), two replacements for some ozone-depleting chlorofluorocarbons (CFC), were investigated in rat liver microsomes and isolated rat hepatocytes. Both compounds were activated by cytochrome P450 to reactive metabolites, as indicated by: (i) the depletion of exogenous and cellular glutathione, (ii) the increased LDH release from hepatocytes, (iii) the loss of microsomal P450 content and activities, and (iv) the formation of free radical species observed in the presence of the two compounds. Moreover, the formation of two stable metabolites and an increased production of conjugated dienes, a marker of lipid peroxidation, were observed for both HCFC-123 and HCFC-141b. The biotransformation of both compounds by pyridine- and phenobarbital-induced rat liver microsomes and the inhibition of LDH release by 4-methylpyrazole and troleandomycin indicate that P450 2E1, 2B and, possibly, also 3A are the isoforms involved in the bioactivation and toxicity of HCFC-123 and HCFC-141b in the rat.

Animals↗

PubMed2001

Hormonal therapy with megestrol in inoperable hepatocellular carcinoma characterized by variant oestrogen receptors.

Variant liver oestrogen receptor transcripts in hepatocellular carcinoma are associated with aggressive clinical course and unresponsiveness to tamoxifen. To evaluate the impact on survival and on tumour growth of megestrol (progestin drug acting at post-receptorial level) we enrolled 45 patients with HCC characterized by variant liver oestrogen receptors in a prospective, randomized study with megestrol vs. placebo. Presence of variant oestrogen receptors was determined by RT/PCR. 24 patients were randomized to no treatment and 21 to therapy with megestrol 160 mg day(-1). Results were analysed by Kaplan-Meier and Cox methods. Survival of hepatocellular carcinoma characterized by variant oestrogen receptors was extremely poor (median survival 7 months); megestrol significantly improved survival (18 months) (P = 0.0090). Tumour growth at one year was significantly slowed down in megestrol-treated patients (P = 0.0212). Bilirubin levels, presence of portal thrombosis, HBV aetiology and treatment were identified at univariate analysis as factors significantly associated with survival; at multivariate analysis, only megestrol therapy (P = 0.0003), presence of HBV infection (P = 0.0009) and presence of portal vein thrombosis (P = 0.0051) were factors independently related with survival. (1) Megestrol slows down the aggressive tumour growth of patients with hepatocellular carcinoma characterized by variant estrogen receptors and (2) is also able to favourably influence the course of disease, more than doubling median survival.

Alternative Splicing↗

PubMed2001

Interaction of processes on different length scales in a bioelastomer capable of performing energy conversion.

This work concerns the aggregation properties of (Gly-Val-Gly-Val-Pro)(251) rec, a polypentapeptide reflecting a highly conserved repetitive unit of the bioelastomer, elastin. On raising the temperature of aqueous solutions above 25 degrees C, this polypeptide was already known to undergo concurrent conformational changes (hydrophobic folding), phase separation, and self-assembly with formation of aggregated three-stranded filaments composed of dynamic polypeptide helices, called beta-spirals. Aggregates obtained from the solution can be shaped into bands that acquire entropic elastic properties upon gamma-irradiation and can perform a variety of energy conversions. Previous studies have shown that aggregation is prompted by the (diverging) critical fluctuations of concentration occurring in the solution, in vicinity of its spinodal line. Here, we present combined circular dicroism (CD) and light scattering experiments, and independent fittings of experimental data to the theoretical spinodal and binodal (coexistence) lines. Results show the following logical and causal sequence of processes: (a) Smooth and progressive conformational changes promoted by concentration fluctuations occurring as temperature is raised "pull down" (in the temperature scale) the instability region of the solution. (b) This further promotes critical fluctuations. (c) The related locally high concentration prompts a further substantial conformational change ending in triple-helix formation and coacervation. (d) This intertwining of processes, covering different length scales (from that of individual peptides to the mesoscopic one of demixed regions), is related to the fact that solvent-induced interactions play a strong role over the entire scale span. These results concur with other recent ones in pointing out that process interactions over many length-scales probably reflect a frequent if not ubiquitous pattern in protein aggregation. This may be highly relevant to the desirable deep understanding of such phenomenon, whose interests cover many fields.

Animals↗

PubMed2001

Bioactivation and cytotoxicity of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) in isolated rat hepatocytes.

The bioactivation and cytotoxicity of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123), a replacement for some ozone-depleting chlorofluorocarbons, were investigated using freshly isolated hepatocytes from non-induced male rats. A time- and concentration-dependent increase in the leakage of lactate dehydrogenase and a concentration-dependent loss of total cellular glutathione were observed in cells incubated with 1, 5 and 10 mM HCFC-123 under normoxic or hypoxic (about 4% O2) conditions. Lactate dehydrogenase leakage was completely prevented by pretreating the cell suspension with the free radical trapper N-t-butyl-alpha-phenylnitrone. The aspecific cytochrome P450 (P450) inhibitor, metyrapone, totally prevented the lactate dehydrogenase leakage from hepatocytes, while two isoform-specific P450 inhibitors, 4-methylpyrazole and troleandomycin (a P450 2E1 and a P450 3A inhibitor, respectively), provided a partial protection against HCFC-123 cytotoxicity. Interestingly, pretreatment of cells with glutathione depletors, such as phorone and diethylmaleate, did not enhance the HCFC-123-dependent lactate dehydrogenase leakage. Two stable metabolites of HCFC-123, 1-chloro-2,2,2-trifluoroethane and 1-chloro-2,2-difluoroethene, were detected by gas chromatography/mass spectrometry analysis of the head space of the hepatocyte incubations carried out under hypoxic and, although at a lower level, also normoxic conditions, indicating that reductive metabolism of HCFC-123 by hepatocytes had occurred. The results overall indicate that HCFC-123 is cytotoxic to rat hepatocytes under both normoxic and hypoxic conditions, due to its bioactivation to reactive metabolites, probably free radicals, and that P450 2E1 and, to a lower extent, P450 3A, are involved in the process.

Animals↗

PubMed2001

Bioactivation to free radicals and cytotoxicity of 1,1-dichloro-1-fluoroethane (HCFC-141b).

1. The in vitro bioactivation by rat liver microsomes and the cytotoxicity in rat hepatocytes of 1,1-dichloro-1-fluoroethane (HCFC-141b), a replacement for some ozone depleting chlorofluorocarbons (CFC), have been investigated. 2. Anaerobic incubations of liver microsomes from pyridine-induced rats with HCFC-141b in the presence of the spin-trapping agent N-t-butyl-alpha-phenylnitrone (PBN) resulted in the formation of a typical ESR radical signal. 3. In the presence of HCFC-141b, a dose-dependent formation of conjugated dienes was observed that was partially inhibited by PBN, glutathione (GSH) and vitamin C. Moreover, HCFC-141b increased the release of lactate dehydrogenase (LDH) and the depletion of cellular glutathione in isolated rat hepatocytes under both normoxic and hypoxic conditions. 4. HCFC-141b-dependent cytotoxicity was completely prevented by PBN under both conditions and it was partially prevented under normoxic conditions by the broad-spectrum P450 inhibitor metyrapone, the P4502E1 specific inhibitor 4-methylpyrazole and the P4503A-specific inhibitor troleandomycin. Interestingly, HCFC-141b-dependent glutathione depletion was not prevented by PBN, metyrapone, 4-methylpyrazole or troleandomycin, whereas two glutathione depletors, 2,6-dimethyl-2,5-heptadien-4-one (phorone) and diethylmaleate, partially prevented LDH release. 5. The present results indicate that HCFC-141b is reductively metabolized in vitro to free radical intermediates by P450, in particular by the CYP2E1 and, to a lower extent, CYP3A isoforms, leading to peroxidative membrane damage and glutathione-independent cytotoxicity.

Animals↗

PubMed2001

Health care utilization and workplace interventions for neck and upper limb problems among newspaper workers.

Data on the use of various therapeutic interventions among working populations at risk for musculoskeletal disorders are rare, despite the need for such information in assessing adherence to best practices. Using the results of a cross-sectional survey of newspaper workers who reported neck and upper limb pain or discomfort (n = 309), we describe the prevalence of a wide range of clinical and workplace interventions. Information/education, exercises, and physical treatments were the most common interventions, and work changes were less prevalent. Those with more frequent, longer-duration, and/or more severe symptoms more commonly reported visits to physiotherapists and health practitioners at work and use of physical treatments, medications, and devices. The multiplicity of interventions used pose evaluation challenges for occupational health practitioners and researchers.

Adult↗

PubMed2001

Genetic repeat polymorphism in the regulating region of CYP2E1: frequency and relationship with enzymatic activity in alcoholics.

BACKGROUND: Differences in the regulatory region of the CYP2E1 gene could be responsible for the interindividual variation in the cytochrome P-450 2E1 (CYP2E1) involved in ethanol oxidation. Recently, a polymorphic repeat sequence in the human gene was described between -2178 and -1945 base pairs. Its frequency seemed to vary among different ethnic populations, and it was suspected to be related to an increased inducibility to further ethanol intake. In the study reported here, the frequency of this polymorphism was investigated in a white French population. Its relationship with the previously described PstI/RsaI or DraI CYP2E1 polymorphisms, alcoholism, alcoholic liver disease, and inducibility of CYP2E1 by ethanol was examined. METHODS: The polymorphic region was characterized by polymerase chain reaction in 103 controls, 148 alcoholic subjects without liver diseases, and 98 others with liver cirrhosis. By using in vivo chlorzoxazone (CHZ) metabolism, CYP2E1 phenotype was assessed in 36 non-ethanol-induced subjects (17 controls and 19 withdrawn alcoholics) and in 14 ethanol-induced subjects (10 controls after ingestion of 0.8 g/kg ethanol and four alcoholics with 100 g of daily intake). This phenotype was expressed as the 6-hydroxy CHZ/CHZ ratio. RESULTS: The rare allele frequency was found to be 1.58% in whites (n = 349). Neither significant association with alcoholism or alcoholic liver diseases, nor relationship with the PstI/RsaI polymorphism, was observed. But the DraI polymorphism was more frequent among the heterozygous subjects when compared with wild-type homozygous ones (p < 0.05). The CYP2E1 phenotype was similar in wild-type homozygotes and in heterozygotes at the constitutive level, as well as after induction with ethanol. CONCLUSIONS: Our data suggest that CYP2E1 repeat polymorphism does not seem to constitute a major factor for interindividual differences in CYP2E1 expression and susceptibility to alcohol-related disorders in whites.

Adult↗

PubMed2000

Structural improvement of higher education in environmental toxicology in Latin America and Europe.

Industrial development has resulted in an increased release of chemicals and other agents into the environment, resulting in damage to the environment as well as increasing the risk of adverse effects on human health. Environmental toxicology (ET) is the discipline responsible for assessing the risks to human health and the environment from the effects of new chemicals and those already present in the environment. The development of human resources in toxicology is therefore a priority in both Latin America (LA) and the European Union (EU), although LA professionals are more involved in risk evaluation than in risk assessment compared to their EU colleagues. A solid background in general toxicology will enable those interested in environmental issues to tackle local problems. Moreover, the increasing globalization of markets and, therefore, of the necessary regulations, requires harmonisation of postgraduate programmes to ensure that risk assessment and management related to the environment are dealt with uniformly and by highly qualified scientists. The Inaugural Meeting of the ALFA-OMET Toxicology', a 2-year programme supported by the European Commission, offered the opportunity to discuss a number of these issues. The present status of existing ET courses in the EU and LA and the corresponding professional profiles in the two regions were examined, and a harmonized academic curriculum for a postgraduate professional profiles in the two regions were examined, and a harmonized academic curriculum for a postgraduate course in environmental toxicology was developed. Finally, a course programme for toxicology and a specialization in environmental toxicology designed by a panel of experts was discussed, and its relevance as a model for other specialisation programmes was analysed. Exercises such as those performed by ALFA-OMET may be useful not only in promoting discussion for the implementation of national and international professional registers in LA, but also in encouraging the same, ongoing process in the EU.

Environmental Pollutants↗

PubMed1999

Interacting processes in protein coagulation.

A strong interest is currently focused on protein self-association and deposit. This usually involves conformational changes of the entire protein or of a fragment. It can occur even at low concentrations and is responsible for pathologies such as systemic amyloidosis, Alzheimer's and Prion diseases, and other neurodegenerative pathologies. Readily available proteins, exhibiting at low concentration self-association properties related to conformational changes, offer very convenient model systems capable of providing insight into this class of problems. Here we report experiments on bovine serum albumin, showing that the process of conformational change of this protein towards an intermediate form required for coagulation occurs simultaneously and interacts with two more processes: mesoscopic demixing of the solution and protein cross-linking. This pathway of three interacting processes allows coagulation even at very low concentrations, and it has been recently observed also in the case of a nonpeptidic polymer. It could therefore be a fairly common feature in polymer coagulation/gelation. Proteins 1999;37:116-120.

Alzheimer Disease↗

PubMed1999

Coccygeal fracture, constipation, convulsion, and confusion: a case report of malignant hypertension in a child.

Malignant hypertension is an unusual but well described cause of seizures in pediatrics. It is a medical emergency that must be recognized and emergently treated to prevent morbidity and mortality. In contrast to adults, hypertension in children is usually secondary to an underlying disease process. We present a complex case of hypertensive encephalopathy with seizures as the initial presentation of a pelvic mass, describe the initial work-up and stabilization and present an overview of the literature. Review of the medical literature described only one similar presentation (1). Interestingly, acute symptoms in this patient may have been precipitated by use of an over-the-counter medication.

Antihypertensive Agents↗

PubMed1999

Chlorzoxazone, a selective probe for phenotyping CYP2E1 in humans.

The ability of human cytochromes P450 other than CYP2E1 to catalyse the 6-hydroxylation of chlorzoxazone (6-OH-CHZ) was examined in vitro using human liver microsomal preparations and in vivo using chlorzoxazone as a metabolic probe. Chlorzoxazone 6-hydroxylation activity was significantly correlated with 4-nitrophenol 2-hydroxylase activity and immunodetected CYP2E1 in 14 human liver samples (r = 0.92 and 0.81, P < 0.001, respectively). Conversely, this catalytic activity was not correlated with CYP 3A or CYP1A activities. Diethyldithiocarbamate (DEDTC), a specific CYP2E1 inhibitor, reduced chlorzoxazone 6-hydroxylase activity by 92.3 +/- 7.6% (n = 14 samples) while ketoconazole, a specific CYP3A inhibitor, reduced this activity by 8.6 +/- 6.3% (n = 14). The residual activity following preincubation with DEDTC was significantly correlated with nifedipine oxidation and tamoxifen N-demethylations, both specific to CYP3A (r = 0.76 and 0.68, respectively). Genetically produced pure human CYP2E1 and 3A4 hydroxylated chlorzoxazone with turnover numbers of 19.7 and 0.14 min(-1), respectively. Furthermore, cytochrome b5 stimulated chlorzoxazone 6-hydroxylation. From examination of the relative liver content of CYP2E1 and 3A, it can be asserted that CYP2E1 is the major enzyme involved in chlorzoxazone 6-hydroxylation and that the contribution of CYP3A is very minor. CYP2E1 activity was evaluated by the plasmatic metabolic ratio 6-OH-CHZ/CHZ (CHZ-MR) measured 2 h after ingestion of 500 mg CHZ. Smoker status did not influence the rate of CHZ hydroxylation. The CHZ-MR was 0.30 +/- 0.13 (mean +/- SD) n = 39 non-smokers versus 0.32 +/- 0.15, n = 75 smokers. This result suggests that CYP1A, inducible by cigarette smoking, is not significantly involved in chlorzoxazone hydroxylation. Women exhibited a slightly lower CHZ-MR than men (0.29 +/- 0.15, n = 44 versus 0.34 +/- 0.15 n = 49, respectively). Obesity increased CHZ-MR, especially in non-insulin-dependent diabetic individuals (0.45 +/- 0.21, n = 13 versus 0.30 +/- 0.15, n = 42 control individuals, P = 0.007). Furthermore, exposure of workers to volatile organics in a shoe factory decreased CHZ-MR (0.19 +/- 0.09, n = 10 Mexican workers versus 0.34 +/- 0.12, n = 16 Mexican control individuals, P = 0.001). Concomitant administration of grapefruit juice (known to be an inhibitor of CYP3A4) with chlorzoxazone did not significantly modify the CHZ metabolic ratio: 0.29 +/- 0.1 versus 0.31 +/- 0.1, for nine control individuals without and with grapefruit juice, respectively. In conclusion, all these results demonstrate that chlorzoxazone is a very selective probe for phenotyping CYP2E1 in humans.

Adult↗

PubMed1997

Cytochrome P450 inactivation during reductive metabolism of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) by phenobarbital- and pyridine-induced rat liver microsomes.

The reductive metabolic activation of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123), one of the potential substitutes for the ozone-depleting chlorofluorocarbons and a close structural analogue of the hepatotoxic anesthetic halothane, was investigated in vitro. During incubation of liver microsomes from phenobarbital-(PB) or pyridine-induced (PYR) rats with 0-20 mM HCFC-123 under anaerobic conditions, a dose- and time-dependent depletion of added exogenous glutathione was observed, indicating the formation of reactive metabolites. Under similar incubation conditions, except for the absence of glutathione, 1-chloro-2,2,2-trifluoroethane and 1-chloro-2,2-difluoroethene were detected as products of reductive metabolism of HCFC-123, as previously reported for halothane. As shown previously in our laboratory for halothane, under these conditions HCFC- 123 also caused a statistically significant loss of microsomal cytochrome P450 (P450) as indicated by a decrease of the classical absorption spectrum in the presence of CO. Both glutathione depletion and P450 loss were almost completely prevented by previous saturation of the incubation mixture with CO and were partially prevented by the presence of the free-radical scavenger N-t-butyl-alpha-phenylnitrone or the carbene trapping agent 2,3-dimethyl-2-butene, suggesting that both types of intermediates may be involved. The loss of P450 was associated with a quantitatively similar loss of microsomal heme, as measured by the pyridine hemochromogen reaction, with PB but not with PYR microsomes. Finally, both the P4502E1-specific p-nitrophenol hydroxylase activity in PYR microsomes and the P4502B1/2-specific pentoxyresorufin O-depentylase activity in PB microsomes were significantly inhibited (58 and 53%, respectively) by prior incubation with HCFC-123, suggesting that both isoforms are able to catalyze the activation of this halogenated compound. These results indicate that indeed HCFC-123, like its analogue halothane, is activated reductively to reactive metabolites by at least two P450 isoforms, namely P4502E1 and P4502B1/2. These metabolites, probably free radicals and/or carbene species, may attack the enzyme resulting in modification of the heme group and subsequent loss of catalytic activity.

Animals↗

PubMed1997

HLA-B8,DR3 haplotype affects lymphocyte blood levels.

The number of lymphocytes in the blood is constant, pointing to an effective control of circulating lymphocyte values. The mechanisms of this regulation are uncertain, although it is likely that the number of blood lymphocytes is conditioned by hormones, homing factors and cytokines whose production is at least partly restrained by genetic factors. Particularly genetic factors linked to major histocompatibility complex (MHC) appear to be involved. In human beings a decreased number of blood lymphocytes has been described in healthy subjects carrying the Human Leucocyte Antigens (HLA) haplotype HLA-B8,DR3. In the present study, to inquire into the mechanisms of this lymphocyte decreased number, we have performed an analysis of blood subset values in these subjects. When the absolute values of lymphocytes were analysed according to HLA phenotype, HLA-B8,DR3 positive subjects (N = 26) displayed significantly lower values as compared to HLA-B8,DR3 negative ones (N = 282). The analysis of lymphocyte subpopulations performed by flow cytometry in 72 subjects did not show significant changes in lymphocyte subset percentages between HLA-B8,DR3 positive subjects and negative ones. Thus, the decrease of circulating lymphocytes seems to be due to a reduction of cell number affecting all lymphocyte subsets rather than a single cell subpopulation. The analysis of in vitro spontaneous apoptosis performed by flow cytometry in a smaller sample of subjects showed a significant increase of spontaneous apoptosis in lymphocytes from HLA-B8,DR3 positive individuals suggesting a possible explanation for the deviation from normal lymphocyte count observed in these subjects. However it is intriguing that a decreased number of blood lymphocytes can be observed in healthy HLA-B8,DR3 positive subjects but also in autoimmune diseases linked to this haplotype like systemic lupus erythematosus and insulin-dependent diabetes. Furthermore, in our opinion, this finding is to be kept in mind in evaluating hematological parameters in healthy subjects.

Adult↗

PubMed1996

Potentiation of occupational carbon tetrachloride toxicity by ethanol abuse.

1. Two separate cases of acute occupational poisoning following exposure to vapours of a fire extinguishing liquid are described. Analytical, clinical, pathological and toxicological data leave little doubt that toxicity was due, in both cases, to inhalation of carbon tetrachloride present at high concentrations ( > 15% and 78% by weight, respectively) in the fire extinguishing liquid. 2. Both subjects were admitted to hospital, 4 and 8 days after exposure, respectively, and developed severe hepato-nephrotoxicity with hepatomegaly, increased values of serum transaminases, blood urea nitrogen (BUN), creatinine, gamma-glutamyl-transpeptidase (gamma-GT), bilirubin and uric acid and, finally, anuria. They recovered in about three to four weeks, after several haemodialytic sessions. 3. Interestingly, in both cases the other workers exposed under the same conditions and for the same period of time as the two patients showed no signs or symptoms of toxicity. The reason for the observed different susceptibility to CCl4 is attributable to an abnormally high ethanol abuse by the two workers, as reported in the clinical records and confirmed by their relatives and colleagues (120 and 250 g per day, respectively). Daily ethanol intake by the coworkers was less than 50 g for all subjects. 4. Although the potentiating effect of ethanol on the toxicity of CCl4 is well known in experimental animals, as a result of cytochrome P-450 induction, direct evidence in humans reported in the literature so far was limited by the lack of information, in all published reports, on the presence or absence of effects in non-alcoholic exposed "controls', when these were present.

Adult↗

PubMed1996

Reductive activation of 1,1-dichloro-1-fluoroethane (HCFC-141b) by phenobarbital- and pyridine-induced rat liver microsomal cytochrome P450.

1. During anaerobic reductive incubation of liver microsomes, from either the pyridine- or phenobarbital-treated rat, with 1,1-dichloro-1-fluoroethane (HCFC-141b) in the presence of a NADPH-regenerating system, a time- and dose-dependent formation of reactive metabolites was detected as indicated by a depletion of added exogenous glutathione. 2. A statistically significant, dose-dependent loss of both cytochrome P450 and microsomal haem was also observed under these experimental conditions. Furthermore, a statistically significant decrease of p-nitrophenol hydroxylase and pentoxyresorufin O-depentylase activity was measured in microsomes from the pyridine- and phenobarbital-induced rat, respectively indicating that both P4502E1 and P4502B undergo substrate-dependent inactivation. 3. Both reactive metabolite formation and P450 inactivation were almost completely inhibited by previous bubbling of the incubation mixture with carbon monoxide, indicating that interaction of the substrate with a free and reduced P450 haem iron is required for substrate bioactivation and enzyme loss. 4. The presence in the incubation mixture of the spin-trap N-t-butyl-alpha-phenylnitrone (PBN) and the carbene trap 2,3-dimethyl-2-butene (DMB) largely prevented both glutathione depletion and P450 loss. This suggests that free radical and carbene intermediates formed by the metabolic activation of the substrate are involved in the inactivation of P450 and the loss of its prosthetic haem group.

Alkenes↗

PubMed1995

Suicidal inactivation of haemoproteins by reductive metabolites of halomethanes: a structure-activity relationship study.

Human haemoglobin (Hb), methaemalbumin (MHA) or rat liver microsomal cytochrome P-450 (P-450) were incubated anaerobically at microM concentrations with 1 mM carbon tetrachloride (CCl4), trichlorobromomethane (CCl3Br), chloroform (CHCl3) or methylene chloride (CH2Cl2) in presence of 1 mM sodium dithionite as the reducing agent. At the end of a 5-min incubation, haem was measured by various methods, i.e. binding spectrum with CO, pyridine-haemochromogen haem assay and porphyrin fluorescence, and compared for the four analogues. Statistically significant losses were observed, with all three haemo-protein systems, for CCi3Br, CCl4 and CHCl3, but not CH2Cl2. For Hb, the loss was greater with CCl3Br (haem assay, 63%; porphyrin fluorescence, 48%; CO binding, 24%) than with CCl4 (haem assay, 31%) or CHCl3 (haem assay, 13%). On the other hand, with MHA, CCl4 gave a dramatic loss (haem assay, 88%; porphyrin fluorescence, 83%; CO binding, 67%), which was greater than that observed with CCl3Br (haem assay, 49%; porphyrin fluorescence, 38%; CO binding, 25%). No loss was found with CHCl3. Finally, with microsomes, the inactivation was larger with CCl4 (CO binding, 58%; haem assay, 50%; porphyrin fluorescence, 33%) than with CCl3Br (CO binding, 33%; haem assay, 10%) or CHCl3 (haem assay, 9%; CO binding, 6%). In a separate set of similar experiments, an ion-pairing reverse phase HPLC method showed the formation of substrate-dependent hae-derived products during incubation of CCl3Br with Hb or microsomes, and of CCl4 with Hb. A correlation between potential for free radical formation (CCl3Br > CCl4 > CHCl3 > CH2Cl2) and extent of haem inactivation was observed with all methods for Hb, but not for microsomal P-450 or MHA. The results indicate that these halomethanes may be activated differently by different haemoproteins and suggest that their potential ability to undergo reductive metabolism may not be the only critical factor involved in P-450 haem inactivation by these chemicals.

Animals↗

PubMed1995

Reductive activation of halothane by human haemoglobin results in the modification of the prosthetic haem.

The metabolic activation of halothane by human haemoglobin (Hb) under reducing conditions in vitro is reported. Absolute spectra of sodium dithionite-reduced Hb, recorded during its anaerobic incubation in the presence of the substrate, showed decreasing concentrations of reduced Hb (Hb2+) with time. The loss of Hb2+ was accompanied, although only to some extent, by a concurrent oxidation to methaemoglobin (Hb3+), suggesting that electron transfer from Hb to the substrate had occurred. Reductive halothane metabolism was observed under these conditions as indicated by a dose-dependent inorganic fluoride (F-) production, which was, however, lower than that observed with heated Hb or a water soluble haem preparation (methaemalbumin). A rapid, partial loss of Hb was found upon addition of the substrate to the incubation mixture, as indicated by a decrease of the typical peak at 418 nm in the absolute spectra recorded in the presence of carbon monoxide (CO). This effect was associated with a loss of the Hb prosthetic group, haem, as shown by a decrease of the pyridine-haemochromogen reaction. Both effects were time and dose dependent. The inhibition of the Hb inactivation reaction by adding exogenous CO or the spin trapping agent N-t-butyl-alpha-phenylnitrone (PBN) to the incubation mixture beforehand indicated that (a) a reduced and free haem iron is required by Hb to activate halothane, and (b) the formation of free radical reactive metabolites of halothane is likely to be responsible for Hb inactivation. The mechanism of the reaction may involve the attack of these metabolites on the haem group of Hb, as indicated by the detection, with a reverse-phase ion-pairing HPLC system, of two Hb-derived products showing a typical haem-like absorption spectrum. The present results resemble those obtained recently with carbon tetrachloride (Ferrara et al., Alternatives to Laboratory Animals 21: 57-64, 1993) and suggest a common mechanism of activation of the two polyhalogenated alkanes by Hb.

Biotransformation↗