Increased Cytochrome P450 Research Articles

Resistance to temephos in Anopheles stephensi larvae is associated with increased cytochrome P450 and α-esterase genes overexpression

The widespread use of synthetic insecticides has resulted in the development of insecticide resistance among disease mosquito vector. Resistance development involves several genes, including CYP450 and esterase family genes. In the current study, expression profiles of seven CYP450 genes namely, CYP9M10, CYP9J40, CYP6AA7, CYP9J34, CYTP450 B9, CYP6M2, CYP6Z2 and three esterase genes namely, esterase β1, alpha esterase, KM234962 were investigated in temephos resistant and susceptible in larvae. The third instar larvae of An. stephensi were subjected to insecticide selection with temephos (Tem Res) following the WHO protocol. Our results show CYP6M2,CYP6Z2 and alpha esterase genes are prominent in temephos resistant in larvae. We suggest that P450 genes and esterase genes, α-esterase and esterase β1 are mostly involved in temephos resistance in larval stage. These results suggest that metabolic resistance may be more prevalent in larval mosquito and play an important role in temephos resistance.

Sex-specific Changes in Brain Estrogen Metabolism Induced by Acute Trimethyltin Exposure.

In this study, we investigated sex-specific effects of acute exposure to trimethyltin, a known neurotoxicant on metabolic steroids. We administered intraperitoneally 2.3 mg/kg trimethyltin to 4-week-old male mice and measured the levels of metabolic steroids 24 h after treatment. We also measured mRNA and protein levels of cytochrome P450 1B1 using real-time polymerase chain reaction and western blotting. Cortisol levels in the cortex increased in both sexes following acute trimethyltin exposure. The estradiol levels decreased, and the 4-hydroxyestradiol levels increased only in females. We also observed increased cytochrome P450 1B1 mRNA and protein levels only in the female cortex. Acute trimethyltin exposure induces distinct sex-specific metabolic changes in the brain before significant sexual maturation.

Adenosine deaminases acting on RNA modulate the expression of the human pregnane X receptor

The pregnane X receptor (PXR) is one of the major transcription factors that regulate the expression of different drug-metabolizing enzymes and transporters. Adenosine-to-inosine RNA editing, the most frequent nucleotide conversion on RNA, which is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes, may modulate gene expression and function. Here, we investigated the potential regulation of human PXR expression by adenosine-to-inosine RNA editing. Knockdown of ADAR1 increased PXR mRNA level, and the knockdown of ADAR1 or ADAR2 significantly increased PXR protein level in HepaRG cells. In HepG2 cells, the knockdown of ADAR1 or ADAR2 significantly increased PXR mRNA and protein levels. The increase in the PXR protein by ADAR1 knockdown resulted in increased cytochrome P450 3A4 (CYP3A4) transactivity and CYP3A4 and UDP-glucuronosyltransferase 1A1 (UGT1A1) expression. A reporter assay revealed that the 3′-untranslated region (UTR) of PXR mRNA, especially from +3371 to +3440, is responsible for the ADAR-mediated post-transcriptional control of PXR expression, despite the lack of RNA edited sites in this region. Collectively, we found that PXR is negatively regulated by ADAR1 via an indirect mechanism, which facilitates the degradation of PXR mRNA. We could demonstrate that ADAR1 can cause interindividual variability in hepatic drug metabolism potencies.

Negative Correlation Between Vitamin A and Positive Correlation Between Vitamin E and Inflammation Among Healthy Adults in Korea: Based on the Korea National Health and Nutrition Examination Survey (KNHANES) 2016-2018 7th Edition.

PurposeVitamins exert its effect through different isoforms. The isoform conversion phases involved are affected outside factors. Here, we investigated the correlation between serum retinol, α-tocopherol, and serum inflammatory markers using stratified data acquired from 2016 to 2018 Korea National Health and Nutrition Examination Survey (KNHANES).Materials and MethodsThis study was based on data acquired from the 7th edition (2016–2018) of the Korea National Health and Nutrition Examination Survey, consisting of survey data on smoking and alcohol drinking, serum retinol level, serum α-tocopherol level, high-sensitivity C-reactive protein (hs-CRP), and baseline characteristics.ResultsThere was a negative correlation between serum retinol and hs-CRP in alcohol drinking men. There was a negative correlation between serum retinol and hs-CRP in the alcohol-nonsmoking female group. There was a positive correlation between α-tocopherol and hs-CRP in the nonsmoking and alcohol-drinking group. There was a positive correlation between α-tocopherol and hs-CRP in the nonsmoking and alcohol-drinking female group. There was positive correlation between vitamin A and E and metabolic syndrome. The lowest vitamin A level was observed in subjects with all five metabolic syndrome criteria matched.ConclusionThere was a negative correlation between serum retinol and hs-CRP and positive correlation between α-tocopherol and hs-CRP. Absorption and secretion of serum retinol are affected by inflammation status through retinol-binding protein. Alcohol acts as a competitive inhibitor of vitamin A oxidation through alcohol dehydrogenase and ALDH activity. Smoking causes inflammation and induces reactive oxygen species scavenging system and increases cytochrome p450 levels. These factors may have contributed to the observed findings. Metabolic syndrome subjects increased as the levels of vitamin A and vitamin E increased. Since obesity is inversely related to ALDH activity, we postulate that patients with metabolic syndrome may also have low ALDH activity, especially in the Asian population. Future studies are warranted to study the efficacy of ALDH or ALDH inducers in patients with vitamin A deficiency or metabolic syndrome.

Mechanisms of Increased Indoxacarb Toxicity in Methoxyfenozide-Resistant Cotton Bollworm Helicoverpa armigera (Lepidoptera: Noctuidae).

Indoxacarb is an important insecticide for the selective control of Helicoverpa armigera. It can be bioactivated to the more effective N-decarbomethoxylated indoxacarb (DCJW) by esterases in pests. It was observed that both field and laboratory selected populations of H. armigera showed negative cross-resistance between indoxacarb and methoxyfenozide. The Handan population exhibited moderate resistance to indoxacarb, but was susceptible to methoxyfenozide; the Baoding and Yishui populations exhibited moderate resistance to methoxyfenozide, but they were susceptible to indoxacarb. Moreover, the toxicity of indoxacarb was enhanced 1.83-fold in the laboratory methoxyfenozide-resistant H. armigera, and susceptibility to methoxyfenozide was increased 2.81-fold in the laboratory indoxacarb-resistant H. armigera. In vivo, DCJW concentrations in the susceptible and methoxyfenozide-selected (laboratory methoxyfenozide-resistant) populations were 4.59- and 4.31-fold greater than in the indoxacarb-resistant Handan population 1 h after dosing. After 2 h, the highest concentrations of DCJW and indoxacarb appeared in the methoxyfenozide-selected population. Meanwhile, increased carboxyl esterase (CarE) and decreased glutathione S-transferase (GST) activities were observed in the methoxyfenozide-selected population. However, the indoxacarb-selected (laboratory indoxacarb-resistant) and Handan populations showed a higher disappearance of indoxacarb and DCJW, and the activity of cytochrome P450 mono-oxygenase in these populations were significantly increased. This study showed that the improved toxicity of indoxacarb, as observed in the methoxyfenozide-selected H. armigera, was correlated with increased CarE activity, decreased GST activity, and the in vivo accumulation of indoxacarb and DCJW. The significantly increased cytochrome P450 activity and higher disappearance of indoxacarb and DCJW in indoxacarb-resistant H. armigera resulted in the decreased toxicity of indoxacarb.

Toxicological and molecular profiling of insecticide resistance in a Brazilian strain of fall armyworm resistant to Bt Cry1 proteins.

BackgroundSpodoptera frugiperda, fall armyworm (FAW) is the major pest of maize in Brazil and has readily acquired field resistance to a broad range of synthetic insecticides and to Bacillus thuringiensis (Bt) insecticidal proteins expressed in important crops. This study aims to understand patterns of cross‐resistance in FAW by investigating the toxicological profile of a Bt‐resistant Brazilian strain (Sf_Des) in comparison to a Bt‐susceptible strain (Sf_Bra).ResultsLaboratory bioassays with 15 active substances of nine mode of action classes revealed that Sf_Des has a medium level of resistance to deltamethrin and chlorpyrifos. Very high cross‐resistance was observed among Cry1 toxins, but high susceptibility against Vip3A. Strain Sf_Des exhibited – depending on the substrate – up to 19‐fold increased cytochrome P450 activity in comparison to Sf_Bra. RNA‐Seq data support a major role of P450 enzymes in the detoxification of insecticides because we detected 85 P450 transcripts upregulated in Sf_Des. Quantitative reverse transcription polymerase chain reaction (RT‐qPCR) analysis confirmed that CYP9A‐like and CYP6B39 are significantly upregulated (>200‐fold) in Sf_Des in comparison to Sf_Bra strain. No target‐site mutation linked to pyrethroid resistance was detected, but mutations in the AChE linked to organophosphate resistance were observed in Sf_Des. A Gene Ontology (GO) analysis of differentially expressed genes (DEG) categorized most of them into the biological process category, involved in oxidation–reduction and metabolic processes.ConclusionOur results indicate that multiple/cross‐resistance mechanisms may have developed in the Sf_Des strain to conventional insecticides and Bt insecticidal proteins. The systematic toxicological analysis presented will help to guide recommendations for an efficient resistance management. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Recapitulation of Human Embryonic Heartbeat to Promote Differentiation of Hepatic Endoderm to Hepatoblasts.

Hepatic development requires multiple sequential physicochemical environmental changes in an embryo, and human pluripotent stem cells (hPSCs) allow for the elucidation of this embryonic developmental process. However, the current in vitro methods for hPSC-hepatic differentiation, which employ various biochemical substances, produce hPSC-derived hepatocytes with less functionality than primary hepatocytes, due to a lack of physical stimuli, such as heart beating. Here, we developed a microfluidic platform that recapitulates the beating of a human embryonic heart to improve the functionality of hepatoblasts derived from hepatic endoderm (HE) in vitro. This microfluidic platform facilitates the application of multiple mechanical stretching forces, to mimic heart beating, to cultured hepatic endoderm cells to identify the optimal stimuli. Results show that stimulated HE-derived hepatoblasts increased cytochrome P450 3A (CYP3A) metabolic activity, as well as the expression of hepatoblast functional markers (albumin, cytokeratin 19 and CYP3A7), compared to unstimulated hepatoblasts. This approach of hepatic differentiation from hPSCs with the application of mechanical stimuli will facilitate improved methods for studying human embryonic liver development, as well as accurate pharmacological testing with functional liver cells.

Cross-resistance and biochemical mechanism of resistance to cyantraniliprole in a near-isogenic line of whitefly Bemisia tabaci Mediterranean (Q biotype)

Bemisia tabaci, sweetpotato whitefly, is one notorious insect pest on a series of crops worldwide, and many populations show high resistance to various insecticides. The cyantraniliprole-resistant strain of B. tabaci SX-R (138.4-fold) was obtained by selections with an outdoor-collected cyantraniliprole resistant population. By crossing and repeated backcrossing to a susceptible MED-S strain, the trait of cyantraniliprole resistance from SX-R was moved into MED-S to establish one near-isogenic line (CYAN-R). MED-S and CYAN-R were utilized to build patterns of cross-resistance, CYAN-R strain exhibited 63.317-fold resistance to cyantraniliprole, but no cross-resistance to several other successfully commercialized chemical agents. After that significant inhibition of cyantraniliprole resistance by piperonyl butoxide (PBO) and increased cytochrome P450 (3.4-fold) were observed in CYAN-R strain, indicating putative involvement of P450 in detoxification. Furthermore, five published detoxification-related P450 genes in B. tabaci, CYP4C64, CYP6CM1, CYP6CX1, CYP6CX4, and CYP6DZ7 were selected and expression levels of them were measured for exploring mechanisms of cyantraniliprole resistance. Compare with MED-S, no significant overexpression of the five P450 genes was observed in the CYAN-R strain. Above results could be conductive to study on mechanism of cyantraniliprole resistance and will be very helpful for the management of whitefly.

Hydrostatic pressure regulates CYP1A2 expression in human hepatocytes via a mechanosensitive aryl hydrocarbon receptor-dependent pathway.

Approximately 75% of xenobiotics are primarily eliminated through metabolism; thus the accurate scaling of metabolic clearance is vital to successful drug development. Yet, when data is scaled from in vitro to in vivo, hepatic metabolic clearance, the primary source of metabolism, is still commonly underpredicted. Over the past decades, with biophysics used as a key component to restore aspects of the in vivo environment, several new cell culture settings have been investigated to improve hepatocyte functionalities. Most of these studies have focused on shear stress, i.e., flow mediated by a pressure gradient. One potential conclusion of these studies is that hepatocytes are naturally “mechanosensitive,” i.e., they respond to a change in their biophysical environment. We demonstrate that hepatocytes also respond to an increase in hydrostatic pressure that, we suggest, is directly linked to the lobule geometry and vessel density. Furthermore, we demonstrate that hydrostatic pressure improves albumin production and increases cytochrome P-450 (CYP) 1A2 expression levels in an aryl hydrocarbon-dependent manner in human hepatocytes. Increased albumin production and CYP function are commonly attributed to the impacts of shear stress in microfluidic experiments. Therefore, our results highlight evidence of a novel link between hydrostatic pressure and CYP metabolism and demonstrate that the spectrum of hepatocyte mechanosensitivity might be larger than previously thought.

Baseline susceptibility and biochemical mechanism of resistance to flupyradifurone in Bemisia tabaci

Bemisia tabaci (Gennadius) is one of the world's most devastating crop pests, and many populations show high resistance to various insecticides. To determine B. tabaci susceptibility to flupyradifurone and understand mechanisms of resistance, we conducted field monitoring and cross-resistance, synergism, and metabolic enzyme activity assays with two resistant strains: Wuhan (WH) and Xiangyang (XY). Results showed that most field populations were highly susceptible to flupyradifurone. However, WH and XY populations, with resistance ratios of 21.95- and 29.03-fold, respectively, showed a low level of flupyradifurone resistance but no cross-resistance to imidacloprid or cyantraniliprole. Synergism tests demonstrated that piperonyl butoxide (PBO) and triphenyl phosphate (TPP) significantly inhibited flupyradifurone resistance in the XY strain, while PBO significantly inhibited resistance in the WH strain. Increased cytochrome P450 monooxygenase and esterase activity correlated with flupyradifurone resistance in the two resistant strains, indicating putative involvement of these enzymes in detoxification. By rotating flupyradifurone with other chemical agents that lack cross-resistance, development of resistance could be delayed, making flupyradifurone an effective agent for whitefly management in China.

Administration of nerve growth factor-β to heifers with a pre-ovulatory follicle enhanced luteal formation and function and promoted LH release

The objective of this study was to determine the effects of bovine nerve growth factor-β (NGF) on pre-ovulatory follicle vascular area, LH release, ovulation, and luteal function when administered systemically to heifers. Post-pubertal Holstein heifers (n = 12) received an intravaginal progesterone-releasing device (CIDR) and GnRH agonist (100 μg IM). The CIDR was removed 5 d later, and heifers were given dinoprost (25 mg IM) at CIDR removal and 24 h later, followed by a second dose of GnRH agonist 48 h later. Heifers were randomly assigned to treatments using a cross-over design. For example, heifers assigned to NGF (250 μg reconstituted in 12 mL PBS IM) in replicate 1 were assigned to control (12 mL PBS IM) in replicate 2. Transrectal ultrasonography was performed before treatment and repeated every 4 h up to 32 h to determine the pre-ovulatory follicle diameter, vascular area, and ovulation. Serum samples were obtained to assess LH concentrations during the periovulatory period and every 2 d post-ovulation for measuring progesterone concentrations. A subset of heifers had luteal biopsies performed on days 9 (n = 6 per treatment) and 14 (n = 6 per treatment) post-ovulation to count luteal cell numbers and measure relative mRNA abundance for steroidogenic and angiogenic enzymes and LH receptor. Treatment with NGF increased pre-ovulatory follicle diameter (P = 0.02) and serum LH concentrations (P = 0.03) but did not affect time to ovulation (P = 0.42). Heifers treated with NGF had increased serum progesterone concentrations in the subsequent luteal phase (P = 0.03), but no change in vascular area of the follicle (P = 0.16) or CL (P = 0.20). Heifers treated with NGF had a greater number of small luteal cells (P < 0.01) and a tendency for increased LH receptor (LHR) mRNA abundance in the CL (P = 0.10). There was also increased steroidogenic acute regulatory protein (STAR; P = 0.05) and a tendency for increased cytochrome P450 family 11 (CYP11A1; P = 0.10) mRNA abundance in the CL of NGF-treated heifers. There was decreased prostaglandin E2 synthase (PGES; P = 0.03) and its receptor (PGER; P = 0.05) mRNA abundance and a tendency for decreased cytochrome P450 family 17 subfamily A member 1 (CYP17A1; P = 0.08) and hydroxysteroid 17-beta dehydrogenase (HSD17B; P = 0.06) mRNA abundance in the CL of NGF-treated heifers. Administration of NGF improved CL function in heifers potentially as a result of increased LH release.

Increasing cytochrome P450 enzyme diversity by identification of two distinct cyclodipeptide dimerases.

Genome mining revealed the presence of two cdps-p450 operons in Saccharopolyspora antimicrobica. Heterologous expression, biochemical characterisation and structure elucidation proved that the two P450 enzymes catalyse distinct regio- and stereospecific dimerizations of cyclo-(l-Trp-l-Trp), which significantly expands the repertoire of diketopiperazine-tailoring enzymes. TtpB1 connects the monomers via C3-C3', both from the opposite side of H-11/H-11', while TtpB2 is characterised as the first P450 to mainly catalyse the unusual linkage between N1' and C3 from the H-11 side.

Brain sterol flux mediated by cytochrome P450 46A1 affects membrane properties and membrane-dependent processes.

Cytochrome P450 46A1 encoded by CYP46A1 catalyzes cholesterol 24-hydroxylation and is a CNS-specific enzyme that controls cholesterol removal and turnover in the brain. Accumulating data suggest that increases in cytochrome P450 46A1 activity in mouse models of common neurodegenerative diseases affect various, apparently unlinked biological processes and pathways. Yet, the underlying reason for these multiple enzyme activity effects is currently unknown. Herein, we tested the hypothesis that cytochrome P450 46A1-mediated sterol flux alters physico-chemical properties of the plasma membranes and thereby membrane-dependent events. We used 9-month-old 5XFAD mice (an Alzheimer’s disease model) treated for 6 months with the anti-HIV drug efavirenz. These animals have previously been shown to have improved behavioural performance, increased cytochrome P450 46A1 activity in the brain, and increased sterol flux through the plasma membranes. We further examined 9-month-old Cyp46a1−/− mice, which have previously been observed to have cognitive deficits and decreased sterol flux through brain membranes. Synaptosomal fractions from the brain of efavirenz-treated 5XFAD mice had essentially unchanged cholesterol levels as compared to control 5XFAD mice. However with efavirenz treatment in these mice, there were changes in the membrane properties (increased cholesterol accessibility, ordering, osmotic resistance and thickness) as well as total glutamate content and ability to release glutamate in response to mild stimulation. Similarly, the cholesterol content in synaptosomal fractions from the brain of Cyp46a1−/− mice was essentially the same as in wild-type mice but knockout of Cyp46a1 was associated with changes in membrane properties and glutamate content and its exocytotic release. Changes in Cyp46a1−/− mice were in the opposite direction to those observed in efavirenz-treated versus control 5XFAD mice. Incubation of synaptosomal fractions with the inhibitors of glycogen synthase kinase 3, cyclin-dependent kinase 5, protein phosphatase 1/2 A, and protein phosphatase 2B revealed that increased sterol flux in efavirenz-treated versus control 5XFAD mice affected the ability of all four enzymes to modulate glutamate release. In contrast, in Cyp46a1−/− versus wild-type mice, decreased sterol flux altered the ability of only cyclin-dependent kinase 5 and protein phosphatase 2B to regulate the glutamate release. Collectively, our results support cytochrome P450 46A1-mediated sterol flux as an important contributor to the fundamental properties of the membranes, protein phosphorylation and synaptic transmission. Also, our data provide an explanation of how one enzyme, cytochrome P450 46A1, can affect multiple pathways and processes and serve as a common potential target for several neurodegenerative disorders.

Consumption of baby kale increased cytochrome P450 1A2 (CYP1A2) activity and influenced bilirubin metabolism in a randomized clinical trial

Brassica vegetables may modulate cancer risk by regulation of xenobiotic metabolizing enzymes (XMEs). In a randomized crossover study, the effect of kale consumption on CYP1A2, CYP2A6, XO, and NAT2 activity was determined by urinary caffeine metabolite ratios, UGT1A1 activity by serum bilirubin concentrations, and GSTA protein and GST activity in blood by ELISA. Adults (n = 25) consumed a basal diet supplemented with kale and radish for 14 days or control vegetables. The kale diet increased CYP1A2 activity by 16.4% on day 8 and 15.2% on day 15 compared to control. Conjugated bilirubin was reduced by the kale diet, decreasing from 19.4 to 14.3 to 9.5% of total bilirubin on days 1, 8, and 15, respectively, which may be explained by induction of MRP2. Other XMEs were not affected by diet. The implications of these results for cancer risk will be clarified as the functions of these XMEs become better understood.

Left Ventricular Hypertrophy: Roles of Mitochondria CYP1B1 and Melatonergic Pathways in Co-Ordinating Wider Pathophysiology.

Left ventricular hypertrophy (LVH) can be adaptive, as arising from exercise, or pathological, most commonly when driven by hypertension. The pathophysiology of LVH is consistently associated with an increase in cytochrome P450 (CYP)1B1 and mitogen-activated protein kinases (MAPKs) and a decrease in sirtuins and mitochondria functioning. Treatment is usually targeted to hypertension management, although it is widely accepted that treatment outcomes could be improved with cardiomyocyte hypertrophy targeted interventions. The current article reviews the wide, but disparate, bodies of data pertaining to LVH pathoetiology and pathophysiology, proposing a significant role for variations in the N-acetylserotonin (NAS)/melatonin ratio within mitochondria in driving the biological underpinnings of LVH. Heightened levels of mitochondria CYP1B1 drive the ‘backward’ conversion of melatonin to NAS, resulting in a loss of the co-operative interactions of melatonin and sirtuin-3 within mitochondria. NAS activates the brain-derived neurotrophic factor receptor, TrkB, leading to raised trophic signalling via cyclic adenosine 3′,5′-monophosphate (cAMP)-response element binding protein (CREB) and the MAPKs, which are significantly increased in LVH. The gut microbiome may be intimately linked to how stress and depression associate with LVH and hypertension, with gut microbiome derived butyrate, and other histone deacetylase inhibitors, significant modulators of the melatonergic pathways and LVH more generally. This provides a model of LVH that has significant treatment and research implications.

Endosulfan toxicity in Nile tilapia (Oreochromis niloticus) and the use of lycopene as an ameliorative agent

ObjectivesEndosulfan is a broad-spectrum organochlorine insecticide that has been commercially in use for decades to control insect pests and has been found to pollute the aquatic environment. The current study was carried out to investigate the toxic effects of endosulfan, an organochlorine pesticide, on Nile tilapia (Oreochromis niloticus), a freshwater fish, and the alleviating effects of lycopene on the induced toxicity. MethodsFour treatment groups of fish were investigated (3 replicates of 15 fish for each group): (1) a control group, (2) a group exposed to endosulfan, (3) a group that was fed on a basal diet supplemented with lycopene, and (4) a group that was fed on a basal diet supplemented with lycopene and exposed to endosulfan. The experiment was carried out over a 4-week period. ResultsEndosulfan negatively affected liver function, including liver enzymes and plasma proteins. Endosulfan affected blood parameters of fish and reduced the counts of red blood cells (RBCs) and white blood cells (WBCs), as well as affected immunological parameters. Endosulfan caused oxidative stress, as it decreased the values of antioxidants catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione (GSH), and increased the level of lipid peroxide malondialdehyde (MDA). Additionally, endosulfan increased cytochrome P450 (CYP450) levels, while it decreased glutathione S-transferase (GST) mRNA transcript levels and distorted the normal histological structure of the liver, gills, and spleen of affected fish. Conversely, lycopene partially restored the aforementioned parameters when administered concomitantly with endosulfan. ConclusionThe results showed the beneficial effects of supplementing fish diets with lycopene as a natural antioxidant for ameliorating the toxicity caused by endosulfan.

Toxicity of cadmium to wheat seedling roots in the presence of graphene oxide

The effects of cadmium (Cd) on wheat seedlings in the presence of graphene oxide (GO) were investigated. Parameters evaluated include root morphology, microtubule protein content, cytochrome P450 activity, and the microcellular structure of wheat seedlings. Compared with treatments with Cd or GO in isolation, treatments combining GO and Cd inhibited the total root length, total root surface area, average root diameter, and number of root hairs. GO combined with Cd also increased cytochrome P450 activity and reduced tubulin content. Cotransport of GO-loading Cd entered root tissues and was then transported to the mesophyll cells; this, in turn, triggered damage to cellular structures, including the cell membranes and chloroplast, leading to root blockage and reduced respiratory efficiency, decreased effectiveness of water and nutrient absorption, and ultimate inhibition of wheat growth and development. These effects of GO exposure were also concentration-dependent. The results indicated that GO amplified the phytotoxicity of Cd in wheat seedling roots. Given the worldwide exposure of the environment to Cd contamination, careful consideration should be given to the effects of GO in combination with Cd in agricultural management.

Cytochrome P450 monooxygenases-mediated sex-differential spinosad resistance in house flies Musca domestica (Diptera: Muscidae)

Females developed notably higher resistance than males in a spinosad-resistant house fly strain, however, resistance factors responsible for this phenomenon are poorly understood. In this study, the potential role of cytochrome P450 monooxygenases involved in the sex-differential spinosad resistance in house flies was investigated, using a susceptible strain (SSS) and a spinosad resistant near-isogenic line (N-SRS). Combination of the synergism of spinosad by PBO and increased cytochrome P450 monooxygenase activity in the N-SRS strain implied that cytochrome P450 monooxygenases contributed to spinosad resistance in house flies. Transcriptional levels of eight P450 genes related to insecticide resistance in two genders of the SSS and N-SRS strain were separately evaluated by quantitative real-time PCR. Notably, compared with the corresponding gender of susceptible SSS house flies, CYP4G2 and CYP6A5v2 were overexpressed in resistant N-SRS females, while the expression of these two P450 genes was significantly decreased in resistant N-SRS males. Furthermore, by measuring the expression of CYP4G2 and CYP6A5v2 in female and male house fly populations with different spinosad resistance levels, which were generated from a series of genetic crosses, the genetic linkage between spinosad resistance and P450 gene expression was analyzed. It was found that with increased spinosad resistance, CYP4G2 and CYP6A5v2 were up-regulated in females, while both of them were down-regulated in males, and this suggested their involvement in the female-linked spinosad resistance of house flies. Taken together, our results provide valuable insight into the involvement of cytochrome P450 monooxygenases in the sex-differential spinosad resistance in house flies.

Cytochrome P450-Mediated λ-Cyhalothrin-Resistance in a Field Strain of Helicoverpa armigera from Northeast China.

Resistance to pyrethroid and organophosphate insecticides has been a growing problem in the management of cotton bollworm Helicoverpa armigera (Hübner) populations in the Yangtze River and Yellow River valleys of China, but resistance status and mechanisms of H. armigera populations from northeast China are less documented. In this study, a field strain collected from Shenyang in northeast China (SYR) is up to 16-fold more resistant than a susceptible strain (SS) to λ-cyhalothrin, while the resistance level to phoxim remained low (2.6-fold). Synergist tests and enzymatic assays show that increased cytochrome P450 monooxygenase (P450) activity is the main mechanism for λ-cyhalothrin resistance. Eight out of 10 genes from CYP6 and CYP9 subfamilies were significantly overexpressed in the SYR strain, and CYP6AE11 was the mostly overexpressed P450 (59-fold). These results suggest that overexpression of multiple P450 enzymes contributes to λ-cyhalothrin resistance in the SYR strain of H. armigera from northeast China.

Utilizing proteomic approach to identify nuclear translocation related serine kinase phosphorylation site of GNMT as downstream effector for benzo[a]pyrene

Glycine N-methyltransferase (GNMT) protein is highly expressed in certain tissues, such as liver, pancreas, and prostate. GNMT serves multiple roles which include a methyl group transfer enzyme and a liver tumor suppressor. Benzo(a)pyrene (BaP), a family member of polycyclic aromatic hydrocarbon (PAH), is a known environmental carcinogen found in coal tar, tobacco smoke, barbecued food and incomplete combustion of auto fuel. BaP recruits cytochrome P450 to transform itself into benzo(a)pyrene-7,8-diol-9,10-epoxide (B(a)PDE), which covalently interacts with DNA causing tumorigenesis. BaP can be detoxified through GNMT and induces GNMT translocation into the cellular nucleus. GNMT translocation is accompanied by phosphorylation, but the role of phosphorylation in GNMT remains to be explored. Using liquid chromatography coupled with tandem mass spectrometry, this study identified serine 9 of GNMT as the phosphorylation site upon BaP treatment. When serine 9 was mutated and lost the capability to be phosphorylated, the occurrence of BaP-induced GNMT nuclear translocation was dramatically decreased. Also, this mutant from of GNMT lost the ability of phosphorylation and increased cytochrome P450 1A1 (Cyp1a) expression upon BaP treatment. In addition, protein kinase C (PKC) and c-Jun NH2-terminal kinase (JNK) may be required for such phosphorylation. Further characterization of phosphorylated GNMT for its link to BaP may bring new insights into chemical detoxification.