Changes in immunohistochemical distribution of cytochrome MC-P-448 in the rat liver during acclimation to cold

Immunohistochemical localization of cytochrome MC-P-448 (form of cytochrome P-450 induced by methylcholanthrene) in rat hepatic lobule and the changes in their distribution pattern in response to cold exposure at 4°C were investigated. The distribution of hepatocytes expressing immunoreactivity to cytochrome MC-P-448 was demonstrated with rabbit anti-MC-P-448 serum using a microphotomeasurement system P1 (Nikon). A duration of cold exposure for 1, 2, 3 or 4 weeks at 4°C was applied to study the effect of cold adaptation of cytochrome MC-P-448. In control rats housed at 24°C, hepatocytes showing high immunoreactivity to cytochrome MC-P-448 were located in the centrilobular areas of the hepatic lobules, whereas they disappeared markedly in the 4-week cold-exposed rats. In 1-week and 2-week cold-exposed rats, only a slight decrease in the expression of MC-P-448 positive hepatocytes was observed. These changes were clearly seen by visual inspection of the distribution topography as determined by a microphotomeasurement technique. In conclusion, cytochrome MC-P-448 forms which were predominantly located in centrilobular areas in the hepatic lobule decreased in 4-week cold-exposed rats. This was in contrast to our early report which showed an increasing tendency of cytochrome PB-P-450 forms in 4-week cold-exposed rats.     

Comparison of the effects of inhibitors of cytochrome P-450-mediated reations on human platelet aggregation and arachidonic acid metabolism

Metyrapone and SKF-525A, together with amphenone B, a structural analogue of metyrapone, which are all inhibitors of cytochrome P-450-mediated reactiors, were shown to inhibit the arachidonic acid-induced aggregation of human platelets. Amphenone B, like metyrapone, exhibited a type II (ligand) binding spectrum with rat liver microsomal cytochrome P-450, in contrast to SKF 525A which is a type I (substrate) binding agent. Independently of their type of binding spectra and of their maximum spectral change, however, the affinity of the three compounds for rat liver cytochrome P-450 showed a close proportional correlation with their platelet aggregation inhibitory potency. All three compounds inhibited the formation of [1?¹⁴C]thromboxane B₂ from [1?¹⁴C]arachidonic acid by human platelets aggregated with collagen. The effect of metyrapone on the remaining labelled products suggested that it is a selective thromboxane synthesis inhibitor, while amphenone B exhibited activity reminiscent of cyclo-oxygenase inhibitors. SKF 525A produced complex effects possibly attributable to cyclo-oxygenase inhibition and enhanced lipid peroxidation, since it also enhanced platelet malonaldehyde formation, which the other two compounds inhibited. These data provide further support for a role of cytochrome P-450 in thromboxane synthesis and platelet aggregation.     

Expression of cytochrome P-450lpr Is developmentally regulated and limited to house fly

Expression of house fly cytochrome P-450_lpr was examined using immunoblotting in male and female adult LPR house flies, mixed sex adult house flies at 12 different ages, larvae, and pupae. P-450_lpr was expressed in both male and female adult house flies. P-450_lpr was clearly present in all adult stages examined, was barely detectable in pupae, and could not be detected in larvae. Thus, cytochrome P-450_lpr is developmentally regulated and present in both sexes of house fly. Expression of cytochrome P-450, immunologically homologous to house fly cytochrome P-450_lpr was examined in other species using immunoblot analysis. Eleven animal species were tested in the orders Diptera, Hymenoptera, Lepidoptera, Orthoptera, Acari, and Rodentia, using microsomes in some species from both induced and noninduced animals or insecticide-resistant and susceptible strains. P-450_lpr appears to be restricted to house flies, as none of these species contained cytochrome P-450 that reacted with antiserum to cytochrome P-450_lpr.     

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.     

昆虫抗药性机理：行为和生理改变及解毒代谢增强（英文）

杀虫剂抗性是指“生物的一个品系发展了对该生物正常种群中大多数个体具有致死作用剂量的杀虫药剂的能力”。行为改变、生理学上的变化或代谢解毒等抗性机制能够降低毒物到达靶标的有效剂量。行为抗性是指减少昆虫与毒物接触或使昆虫能够存活于对大多数对正常个体致死（或有害）的环境中的任何行为。生理学改变的机制包括杀虫剂对表皮的穿透性降低、增加对药剂阻隔（sequestration）或储存和加速杀虫剂的排泄。细胞色素P450、水解酶和谷胱甘肽S-转移酶是杀虫药剂代谢解毒的主要3大酶系。细胞色素P450是一个超基因家族,是生物体内对外源性和内源性化合物解毒代谢或活化最重要的酶系。在许多害虫中发现P450介导的解毒代谢增加导致了对杀虫药剂抗性的增加。谷胱甘肽S-转移酶是可溶性的 二聚体蛋白,与代谢解毒、大量内源性和外源性化合物的排泄有关,许多昆虫中证明其抗药性与该酶活性增加有关。水解酶实际上是一组异源的酶类,其对抗药性的作用包括通过基因扩增增加酶量,作为结合蛋白隔离杀虫药剂或通过增加酶的活性加强对药剂的水解作用。     

Relative contribution of detoxifying enzymes to pyrethroid resistance in a resistant strain of Helicoverpa armigera

Abstract:  The relative contribution of oxidases and esterases to pyrethroid resistance was studied in a YS-FP strain of Helicoverpa armigera from China. The YS-FP strain was derived from a field-collected strain (YS) by 16 generations of selection with a mixture of fenvalerate and phoxim. Compared with the YS strain, the YS-FP strain showed 1850- to >7140-fold resistance to four ester-bonded phenoxybenzyl alcohol pyrethroids (fenvalerate, deltamethrin, cypermethrin and cyhalothrin), >205-fold resistance to a non-ester phenoxybenzyl alcohol pyrethroid (etofenprox) and only 19-fold resistance to an ester-bonded methylated biphenyl alcohol pyrethroid (bifenthrin). The oxidase inhibitor piperonyl butoxide eliminated most the of resistance to fenvalerate, deltamethrin, cypermethrin, cyhalothrin and etofenprox, whereas the esterase inhibitor S,S,S -tributylphosphorothioate had a small synergistic effect for fenvalerate and cyhalothrin only. This suggests that the resistance to these pyrethroids in the YS-FP strain was mainly because of enhanced oxidative detoxification. The monooxygenase activities of the midguts of sixth-instar larvae of the YS-FP strain to substrates p -nitroanisole, ethoxycoumarin and methoxycoumarin were 3.7-, 4.7- and 10-fold, respectively, compared with that of the YS strain. Glutathione S -transferase activity and esterase activity were not significantly altered in the YS-FP strain. This confirms that enhanced oxidative detoxification was a major mechanism contributing to pyrethroid resistance in the YS-FP strain.     

盐藻对除草剂草丁膦的抗性研究

对分离出的单藻落盐藻进行除草剂草丁膦的抗性实验,液体培养结果显示,野生盐藻对除草剂草丁膦敏感,3.0mg/L剂量的草丁膦能完全抑制盐藻的生长和增殖。     

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F₂ populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries.     

Detoxification of chlorotoluron in a chlorotoluron-resistant biotype of Alopecurus myosuroides. Comparison between cell cultures and whole plants

The metabolism of chlorotoluron in whole plants and cell suspensions was investigated in a previously characterized chlorotoluron-resistant biotype of Alopecurus myosuroides Huds. Both resistant plants and cell suspensions showed a greater capability to metabolize chlorotoluron to non-phytotoxic compounds than the respective susceptible counterparts. Data revealed that although both biotypes degraded chlorotoluron by N -dealkylation and ring-methyl hydroxylation, the resistant biotype showed an enhanced capacity to hydroxylate the parent herbicide. The cytochrome (Cyt) P450 inhibitor 1-aminobenzotriazole (ABT) inhibited the metabolism of chlorotoluron in both resistant and susceptible plants by reducing the formation of non-toxic aryl-hydroxylated derivatives and polar conjugates. N -demethylations were less susceptible to ABT than the other oxidative reactions, but this does not necessarily imply that the second detoxification activity is not Cyt P450, as some P450 activities are more susceptible to ABT than others. Ring-methyl hydroxylation inhibition affected the ability of resistant plants to recover photosynthetic activity after incubation in chlorotoluron, showing a similar fluorescence pattern to susceptible plants in the same conditions without ABT. Fluorescence and metabolism data strongly support the thesis of Cyt P450-mediated 4-methylphenyl hydroxylation as the main route of detoxification of chlorotoluron in the resistant biotype.     

Rhodnius prolixus supergene families of enzymes potentially associated with insecticide resistance

Chagas disease or American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi. Once known as an endemic health problem of poor rural populations in Latin American countries, it has now spread worldwide. The parasite is transmitted by triatomine bugs, of which Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae) is one of the vectors and a model organism. This species occurs mainly in Central and South American countries where the disease is endemic. Disease prevention focuses on vector control programs that, in general, rely intensely on insecticide use. However, the massive use of chemical insecticides can lead to resistance. One of the major mechanisms is known as metabolic resistance that is associated with an increase in the expression or activity of detoxification genes. Three of the enzyme families that are involved in this process – carboxylesterases (CCE), glutathione s-transferases (GST) and cytochrome P450s (CYP) – are analyzed in the R. prolixus genome. A similar set of detoxification genes to those of the Hemipteran Acyrthosiphon pisum but smaller than in most dipteran species was found in R. prolixus genome. All major CCE classes (43 genes found) are present but the pheromone/hormone processing class had fewer genes than usual. One main expansion was detected on the detoxification/dietary class. The phosphotriesterase family, recently associated with insecticide resistance, was also represented with one gene. One microsomal GST gene was found and the cytosolic GST gene count (14 genes) is extremely low when compared to the other hemipteran species with sequenced genomes. However, this is similar to Apis mellifera, a species known for its deficit in detoxification genes. In R. prolixus 88 CYP genes were found, with representatives in the four clans (CYP2, CYP3, CYP4 and mitochondrial) usually found in insects. R. prolixus seems to have smaller species-specific expansions of CYP genes than mosquitoes and beetles, among others. The number of R. prolixus CYP genes is similar to the hemipteran Ac. pisum, although with a bigger expansion in CYP3 and CYP4 clans, along with several gene fragments, mostly in CYP4 clan. Eleven founding members of new families were detected, consisting of ten genes in the CYP3 clan and 1 gene in the CYP4 clan. Members of these clans were proposed to have important detoxification roles in insects. The identification of CCE, GST and CYP genes is of utmost importance for directing detoxification studies on triatomines that can help insecticide management strategies in control programs.     

Induced tolerance of neonate Heliothis zea to host plant allelochemicals and carbaryl following incubation of eggs on foliage of Lycopersicon hirsutum f. glabratum

Summary Incubation of Heliothis zea (Boddie) eggs on foliage of Lycopersicon hirsutum f. glabratum C.H. Mull (accession PI 134417) results in neonates with elevated levels of tolerance to the toxic effects of PI 134417 foliage attributable to 2-tridecanone found in the glandular trichomes which abound on that foliage. The neonates from such eggs are also shown to have elevated levels of tolerance to the carbamate insecticide carbaryl. Incubation of eggs in an atmosphere containing 2-tridecanone similarly produced elevated levels of tolerance to 2-tridecanone among resulting neonates, indicating that 2-tridecanone is the likely inducing agent and that exposure to 2-tridecanone vapor, which is known to emanate from PI 134417 foliage, is sufficient for induction. Analysis of the cytochrome P-450 content in gut microsomes of fifth instar larvae indicated that exposure of larvae to 2-tridecanone in artificial diet or to PI 134417 foliage resulted in significantly elevated levels of cytochrome P-450 relative to larvae fed diet without 2-tridecanone or foliage of L. esculentum which contains no 2-tridecanone. In addition, removal of the glandular trichomes from PI 134417 foliage eliminated the ability of that foliage to induce elevated levels of cytochrome P-450. These results provide circumstantial evidence that cytochrome P-450 may be involved in the induced tolerance to xenobiotics among neonates from eggs exposed to 2-tridecanone or PI 134417 foliage.Support for this research was provided by the USDA Competitive Research Grants Program in Biological Stress under Grant No. 83-CRCR-1-1241 and Grant No. 85-CRCR-1-1615, and the North Carolina Agricultural Research Service. Paper No. 10856 of Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC, USA 27650. Use of trade names does not imply endorsement of products named nor criticisms of similar ones not mentioned     

Cellular Mechanisms of Resistance to Chronic Oxidative Stress

Oxidative stress is implicated in several pathologies such as AIDS, Alzheimer’s disease, and Parkinson’s disease, as well as in normal aging. As a model system to study the response of cells to oxidative insults, glutamate toxicity on a mouse nerve cell line, HT-22, was examined. Glutamate exposure kills HT-22 via a nonreceptor-mediated oxidative pathway by blocking cystine uptake and causing depletion of intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species and, ultimately, apoptotic cell death. Several HT-22 subclones that are 10-fold resistant to exogenous glutamate were isolated and the mechanisms involved in resistance characterized. The expression levels of neither heat shock proteins nor apoptosis-related proteins are changed in the resistant cells. In contrast, the antioxidant enzyme catalase, but not glutathione peroxidase nor superoxide dismutase, is more highly expressed in the resistant than in the parental cells. In addition, the resistant cells have enhanced rates of GSH regeneration due to higher activities of the GSH metabolic enzymes γ-glutamylcysteine synthetase and GSH reductase, and GSH S-transferases activities are also elevated. As a consequence of these alterations, the glutamate resistant cells are also more resistant to organic hydroperoxides and anticancer drugs that affect these GSH enzymes. These results indicate that resistance to apoptotic oxidative stress may be acquired by coordinated changes in multiple antioxidant pathways.     

Localization of Drug-Metabolizing Enzyme Activities to Blood-Brain Interfaces and Circumventricular Organs

Abstract: The brain, with the exception of the choroid plexuses and Circumventricular organs, is partially protected from the invasion of blood-borne chemicals by the specific morphological properties of the cerebral micro-vessels, namely, the tight junctions of the blood-brain barrier. Recently, several enzymes that are primarily involved in hepatic drug metabolism have been shown to exist in the brain, albeit at relatively low specific activities. In the present study, the hypothesis that these enzymes are located primarily at blood-brain interfaces, where they form an "enzymatic barrier," is tested. By using microdissection techniques or a gradient-centrifugation isolation procedure, the activities of seven drug-metabolizing enzymes in isolated microvessels, choroid plexuses, meningeal membranes, and tissue from three Circumventricular organs (the neural lobe of the hypophysis, pineal gland, and median eminence) were assayed. With two exceptions, the activities of these enzymes were higher in the three Circumventricular organs and cerebral microvessel than in the cortex. Very high membrane-bound epoxide hydrolase and UDP-glucuronosyltransferase activities (approaching those in liver) and somewhat high 7-benzoxyre-sorufin- O -dealkylase and NADPH-cytochrome P-450 reductase activities were determined in the choroid plexuses. The pia-arachnoid membranes, but not the dura matter, displayed drug-metabolizing enzyme activities, notably that of epoxide hydrolase: The drug-metabolizing enzymes located at these nonparenchymal sites may function to protect brain tissue from harmful compounds.     

Resistance cost of a cytochrome P450 herbicide metabolism mechanism but not an ACCase target site mutation in a multiple resistant Lolium rigidum population

Costs of resistance are predicted to reduce plant productivity in herbicide-resistant weeds. Lolium rigidum herbicide-susceptible individuals (S), individuals possessing cytochrome P450-based herbicide metabolism (P450) and multiple resistant individuals possessing a resistant ACCase and enhanced cytochrome P450 metabolism (ACCase/P450) were grown in the absence of mutual plant interaction to estimate plant growth traits. Both P450 and ACCase/P450 resistant phenotypes produced less above-ground biomass than the S phenotype during the vegetative stage. Reduced biomass production in the resistant phenotypes corresponded to a reduced relative growth rate and a lower net assimilation rate and rate of carbon fixation. There were no significant differences between the two resistant phenotypes, suggesting that costs of resistance are associated with P450 metabolism-based resistance. There were no differences in reproductive output among the three phenotypes, indicating that the cost of P450 resistance during vegetative growth is compensated during the production of reproductive structures. The P450-based herbicide metabolism is shown to be associated with physiological resistance costs, which may be manipulated by agronomic management to reduce the evolution of herbicide resistance.     

Prediction of Antibiotic Resistance Evolution by Growth Measurement of All Proximal Mutants of Beta-Lactamase

The antibiotic resistance crisis continues to threaten human health. Better predictions of the evolution of antibiotic resistance genes could contribute to the design of more sustainable treatment strategies. However, comprehensive prediction of antibiotic resistance gene evolution via laboratory approaches remains challenging. By combining site-specific integration and high-throughput sequencing, we quantified relative growth under the respective selection of cefotaxime or ceftazidime selection in ∼23,000 Escherichia coli MG1655 strains that each carried a unique, single-copy variant of the extended-spectrum β-lactamase gene bla_CTX-M-14 at the chromosomal att HK022 site. Significant synergistic pleiotropy was observed within four subgenic regions, suggesting key regions for the evolution of resistance to both antibiotics. Moreover, we propose PEAR^P and PEAR^R, two deep-learning models with strong clinical correlations, for the prospective and retrospective prediction of bla_CTX-M-14 evolution, respectively. Single to quintuple mutations of bla_CTX-M-14 predicted to confer resistance by PEAR^P were significantly enriched among the clinical isolates harboring bla_CTX-M-14 variants, and the PEAR^R scores matched the minimal inhibitory concentrations obtained for the 31 intermediates in all hypothetical trajectories. Altogether, we conclude that the measurement of local fitness landscape enables prediction of the evolutionary trajectories of antibiotic resistance genes, which could be useful for a broad range of clinical applications, from resistance prediction to designing novel treatment strategies.     

Cross-induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: impact on larval tolerance to chemical insecticides

The effect of exposure of Aedes aegypti larvae to sub-lethal doses of the pyrethroid insecticide permethrin, the organophosphate temephos, the herbicide atrazine, the polycyclic aromatic hydrocarbon fluoranthene and the heavy metal copper on their subsequent tolerance to insecticides, detoxification enzyme activities and expression of detoxification genes was investigated. Bioassays revealed a moderate increase in larval tolerance to permethrin following exposure to fluoranthene and copper while larval tolerance to temephos increased moderately after exposure to atrazine, copper and permethrin. Cytochrome P450 monooxygenases activities were induced in larvae exposed to permethrin, fluoranthene and copper while glutathione S-transferase activities were induced after exposure to fluoranthene and repressed after exposure to copper. Microarray screening of the expression patterns of all detoxification genes following exposure to each xenobiotic with the Aedes Detox Chip identified multiple genes induced by xenobiotics and insecticides. Further expression studies using real-time quantitative PCR confirmed the induction of multiple CYP genes and one carboxylesterase gene by insecticides and xenobiotics. Overall, this study reveals the potential of xenobiotics found in polluted mosquito breeding sites to affect their tolerance to insecticides, possibly through the cross-induction of particular detoxification genes. Molecular mechanisms involved and impact on mosquito control strategies are discussed.