Arachidonic acid metabolites of CYP4A and CYP4F are altered in women with preeclampsia.

Few studies exist on cytochrome P450 (CYP450) metabolites of arachidonic acid (AA) pertaining to the pathophysiological events in pregnancy. We hypothesized that metabolism of AA via the CYP450 pathways is altered within the placenta in women with preeclampsia (PE) and contributes to the pathophysiology of the disease. Thus, placental vascular CYP450 enzyme expression and activity were measured in normal pregnant (NP) and preeclamptic (PE) patients. CYP450 isoform expression (CYP4A11, CYP4A22, CYP4F2, and CYP4F3) was found to be elevated within the placenta of women with PE compared to normal pregnant (NP) women and chronic hypertensive (CHTN) pregnant women. In addition, placental production of 20-HETE was significantly increased in PE women compared to both NP and CHTN women. Moreover, there was an imbalance in circulating 20-HETE:EETs in PE women. To examine whether alterations in CYP450 AA metabolism contribute to the altered placentation in PE, trophoblast function, proliferation and migration were assessed in the presence of exogenous 20-HETE and a 20-HETE specific synthesis inhibitor, HET0016. Trophoblast proliferation was significantly increased in the presence of 20-HETE (1 µM) and reduced with 20-HETE blockade by HET0016 (1 mM, 5 mM, and 10 mM). On the contrary, administration of exogenous 20-HETE (1 µM) significantly reduced trophoblast migration. In conclusion, metabolism of AA via CYP450 is altered in PE, and increased placental production of 20-HETE may contribute to the pathophysiology of the disease.

Production and characterization of novel hydrocarbon degrading enzymes from Alcanivorax borkumensis.

This study investigates the production of alkane hydroxylase, lipase and esterase by the marine hydrocarbon degrading bacteria Alcanivorax borkumensis. The focus of this study is the remediation of petroleum hydrocarbons, hexane, hexadecane and motor oil as model substrates. A. borkumensis showed an incremental growth on these substrates with a high cell count. Growth on motor oil showed highest alkane hydroxylase and lipase production of 2.62 U/ml and 71 U/ml, respectively, while growth on hexadecane showed the highest esterase production of 57.5 U/ml. The percentage of hexane, hexadecane, and motor oil degradation during A. borkumensis growth after 72 h, was around 80%, 81.5% and 75%, respectively. Zymogram showed two different bands with a molecular weight of approx. 52 and 40 kDa, respectively with lipase and esterase activity. Alkane hydroxylase reached optimum activity at pH 8.0 and 70 ±â€¯1 °C for hexane and hexadecane and 75 ±â€¯1 °C for motor oil. Lipase and esterase showed optimum activity at 35 ±â€¯1 °C and 40 ±â€¯1 °C, respectively and pH 7.0. The crude enzymes showed higher stability in a wide range of pH, but they were not thermostable at higher temperatures.

Association study and expression analysis of CYP4A11 gene copy number variation in Chinese cattle.

The identification of copy number variations (CNVs) allow us to explore genomic polymorphisms. In recent years, significant progress in understanding CNVs has been made in studies of human and animals, however, association and expression studies of CNVs are still in the early stage. It was previously reported that the Cytochrome P-450 4A11 (CYP4A11) gene is located within a copy number variable region (CNVR) that encompasses quantitative trait loci (QTLs) for economic traits like meat quality and milk production. So, this study was performed to determine the presence of CYP4A11 CNV in six distinct cattle breeds, identify its relationship with growth, and explore the biological effects of gene expression. For three CYP4A11 CNV types, Normal was more frequent than Gain or Loss. Association analysis revealed a positive effect of CYP4A11 copy number on growth traits (P < 0.05). One-way analysis of variance (ANOVA) analysis revealed that more CYP4A11 copies increased the gene expression level. Moreover, overexpression of CYP4A11 in vitro revealed its effect on lipid deposit. The data provide evidence for the functional role of CYP4A11 CNV and provide the basis for future applications in cattle breeding.

20-Hydroxyeicosatetraenoic Acid (HETE)-dependent Hypertension in Human Cytochrome P450 (CYP) 4A11 Transgenic Mice: NORMALIZATION OF BLOOD PRESSURE BY SODIUM RESTRICTION, HYDROCHLOROTHIAZIDE, OR BLOCKADE OF THE TYPE 1 ANGIOTENSIN II RECEPTOR.

Male and female homozygous 129/Sv mice carrying four copies of the human cytochrome P450 4A11 gene (CYP4A11) under control of its native promoter (B-129/Sv-4A11(+/+)) develop hypertension (142 ± 8 versus 113 ± 7 mm Hg systolic blood pressure (BP)), and exhibit increased 20-hydroxyeicosatetraenoic acid (20-HETE) in kidney and urine. The hypertension is reversible by a low-sodium diet and by the CYP4A inhibitor HET0016. B-129/Sv-4A11(+/+) mice display an 18% increase of plasma potassium (p < 0.02), but plasma aldosterone, angiotensin II (ANGII), and renin activities are unchanged. This phenotype resembles human genetic disorders with elevated activity of the sodium chloride co-transporter (NCC) and, accordingly, NCC abundance is increased by 50% in transgenic mice, and NCC levels are normalized by HET0016. ANGII is known to increase NCC abundance, and renal mRNA levels of its precursor angiotensinogen are increased 2-fold in B-129/Sv-4A11(+/+), and blockade of the ANGII receptor type 1 with losartan normalizes BP. A pro-hypertensive role for 20-HETE was implicated by normalization of BP and reversal of renal angiotensin mRNA increases by administration of the 20-HETE antagonists 2-((6Z,15Z)-20-hydroxyicosa-6,15-dienamido)acetate or (S)-2-((6Z,15Z)-20-hydroxyicosa-6,15-dienamido)succinate. SGK1 expression is also increased in B-129/Sv-4A11(+/+) mice and paralleled increases seen for NCC. Losartan, HET0016, and 20-HETE antagonists each normalized SGK1 mRNA expression. These results point to a potential 20-HETE dependence of intrarenal angiotensinogen production and ANGII receptor type 1 activation that are associated with increases in NCC and SGK1 and identify elevated P450 4A11 activity and 20-HETE as potential risk factors for salt-sensitive human hypertension by perturbation of the renal renin-angiotensin axis.

Inhibition of CYP4A reduces hepatic endoplasmic reticulum stress and features of diabetes in mice.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus. ER stress activates the unfolded protein response pathway, which contributes to apoptosis and insulin resistance. We investigated the roles of cytochrome P450 4A (CYP4A) in the regulation of hepatic ER stress, insulin resistance, and the development of diabetes in mice. METHODS: We used mass spectrometry to compare levels of CYP450 proteins in livers from C57BL/6J and C57BL/KsJ-db/db (db/db) mice; findings were confirmed by immunoblot and real-time PCR analyses. To create a model of diet-induced diabetes, C57BL/6J mice were placed on high-fat diets. Mice were given intraperitoneal injections of an inhibitor (HET0016) or an inducer (clofibrate) of CYP4A, or tail injections of small hairpin RNAs against CYP4A messenger RNA; liver tissues were collected and analyzed for ER stress, insulin resistance, and apoptosis. The effect of HET0016 and CYP4A knockdown also were analyzed in HepG2 cells. RESULTS: Levels of the CYP4A isoforms were highly up-regulated in livers of db/db mice compared with C57BL/6J mice. Inhibition of CYP4A in db/db and mice on high-fat diets reduced features of diabetes such as insulin hypersecretion, hepatic steatosis, and increased glucose tolerance. CYP4A inhibition reduced levels of ER stress, insulin resistance, and apoptosis in the livers of diabetic mice; it also restored hepatic functions. Inversely, induction of CYP4A accelerated ER stress, insulin resistance, and apoptosis in livers of db/db mice. CONCLUSIONS: CYP4A proteins are up-regulated in livers of mice with genetically induced and diet-induced diabetes. Inhibition of CYP4A in mice reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis. Strategies to reduce levels or activity of CYP4A proteins in liver might be developed for treatment of patients with type 2 diabetes.

Influence of fat/carbohydrate ratio on progression of fatty liver disease and on development of osteopenia in male rats fed alcohol via total enteral nutrition (TEN).

Alcohol abuse is associated with the development of fatty liver disease and also with significant osteopenia in both genders. In this study, we examined ethanol-induced pathology in response to diets with differing fat/carbohydrate ratios. Male Sprague-Dawley rats were fed intragastrically with isocaloric liquid diets. Dietary fat content was either 5% (high carbohydrate, HC) or 45% (high fat, HF), with or without ethanol (12-13 g/kg/day). After 14, 28, or 65 days, livers were harvested and analyzed. In addition, bone morphology was analyzed after 65 days. HC rats gained more weight and had larger fat pads than HF rats with or without ethanol. Steatosis developed in HC + ethanol (HC + EtOH) compared to HF + ethanol (HF + EtOH) rats, accompanied by increased fatty acid (FA) synthesis and increased nuclear carbohydrate response element binding protein (ChREBP) (p < 0.05), but in the absence of effects on hepatic silent mating type information regulation 2 homolog (SIRT-1) or nuclear sterol regulatory binding element protein (SREBP-1c). Ethanol reduced serum leptin (p < 0.05) but not adiponectin. Over time, HC rats developed fatty liver independent of ethanol. FA degradation was significantly elevated by ethanol in both HC and HF groups (p < 0.05). HF + EtOH rats had increased oxidative stress from 28 days, increased necrosis compared to HF controls and higher expression of cytochromes P450, CYP2E1, and CYP4A1 compared to HC + EtOH rats (p < 0.05). In contrast, HC + EtOH rats had no significant increase in oxidative stress until day 65 with no observed increase in necrosis. Unlike liver pathology, no dietary differences were observed on ethanol-induced osteopenia in HC compared to HF groups. These data demonstrate that interactions between diet composition and alcohol are complex, dependent on the length of exposure, and are an important influence in development of fatty liver injury. Importantly, it appears that diet composition does not affect alcohol-associated skeletal toxicity.

Expression of CYP4 and GSTr genes in Venerupis philippinarum exposed to benzo(a)pyrene.

Bivalve molluscs, such as Venerupis philippinarum, are often used as bioindicators of environmental pollution since they can bioaccumulate a large variety of pollutants because of their filter feeding. The Polycyclic Aromatic Hydrocarbon (PAH) benzo(a)pyrene (B(a)P) is an important contaminant, commonly present in the marine environment. Pollutants are generally metabolized by enzymes of phase I, mainly CYPs enzymes, and by conjugation enzymes of phase II like GST. In this study, we investigated by Real Time PCR the expression of CYP4 and GSTr (GST class rho) in the digestive gland of V. philippinarum exposed to different concentrations of B(a)P for 24 h and after a 24 h depuration period. Accumulation of B(a)P by clams has been confirmed by the HPLC-FLD analyses. Moreover, HPLC-FLD analyses evidenced that after depuration, B(a)P concentrations decreased in animals subjected to 0.03 mg/l and 0.5mg/l exposures but did not decrease in animals subjected to 1mg/l exposure. B(a)P exposure and depuration did not cause histopathological lesions in the different organs. The analysis of GSTr expression in the digestive gland showed a significant increase in mRNA in animals subjected to 1 mg/l exposure, whereas the analysis of CYP4 expression did not evidence differences among treatments. Moreover, the expression of both genes did not exhibit any differences after the purification treatment. The results demonstrate that B(a)P significantly affects the expression of GSTr mRNA in the digestive gland of V. philippinarum and suggest that GSTr gene could play an important role in the biotransformation of B(a)P.

A toolkit to enable hydrocarbon conversion in aqueous environments.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)(9) addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments. A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native ß-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF6(8,21) were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced(10,11). The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed. To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources. The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and ß, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and ß improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed. Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

Arterial carbon dioxide partial pressure influences CYP4A distribution in the rat brain.

PaCO(2) is an important factor in the regulation of cerebral circulation, and it is often used to reduce intracranial pressure through hyperventilation during neurosurgery. Changes in concentration can cause changes in CBF (cerebral blood flow). 20-HETE is a product of CYP4A-mediated AA (arachidonic acid) metabolism and is a powerful endogenous vasoconstrictor; however, its effect on cerebral vasoconstriction in cats, dogs and rats remains to be confirmed. It is known that changes in PaCO(2) can influence the expression of CYP4A in the rat brain, demonstrating the important role of 20-HETE in the mechanism of CO(2)-mediated cerebrovascular reactivity. Thirty healthy adult male Wistar rats that weighed between 200 g and 250 g were randomly divided into three groups (A, B, and C; n=10): group A, normocapnia (PaCO(2) was maintained at approximately 40-45 mmHg); group B, hypocapnia (PaCO(2) was maintained at approximately 20-25 mmHg); and group C, hypercapnia (PaCO(2) was maintained at approximately 60-65 mmHg). Physiological parameters, including HR (heart rate), MBP(mean blood pressure), PH and PaCO(2) were recorded every 30 min, and there were no significant hemodynamic or body temperature differences. The head was removed after 3.5 h to investigate brain CYP4A by immunohistochemistry. Relative to group A, group B exhibited the following changes: an increased pH, decreased PaCO(2), and increased brain CYP4A protein expression (P<0.05). In contrast, group C exhibited decreased PH, increased PaCO(2) and decreased CYP4A protein expression (P<0.05). CO(2) can decrease the expression of brain CYP4A during hypercapnia and increase its expression during hypocapnia.

Increase of 20-HETE synthase after brain ischemia in rats revealed by PET study with 11C-labeled 20-HETE synthase-specific inhibitor.

20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid metabolite known to be produced after cerebral ischemia, has been implicated in ischemic and reperfusion injury by mediating vasoconstriction. To develop a positron emission tomography (PET) probe for 20-HETE synthase imaging, which might be useful for monitoring vasoconstrictive processes in patients with brain ischemia, we synthesized a (11)C-labeled specific 20-HETE synthase inhibitor, N'(4-dimethylaminohexyloxy)phenyl imidazole ([(11)C]TROA). Autoradiographic study showed that [(11)C]TROA has high-specific binding in the kidney and liver consistent with the previously reported distribution of 20-HETE synthase. Using transient middle cerebral artery occlusion in rats, PET study showed significant increases in the binding of [(11)C]TROA in the ipsilateral hemisphere of rat brains after 7 and 10 days, which was blocked by co-injection of excess amounts of TROA (10 mg/kg). The increased [(11)C]TROA binding on the ipsilateral side returned to basal levels within 14 days. In addition, quantitative real-time PCR revealed that increased expression of 20-HETE synthase was only shown on the ipsilateral side on day 7. These results indicate that [(11)C]TROA might be a useful PET probe for imaging of 20-HETE synthase in patients with cerebral ischemia.

Cloning and expression of alkane hydroxylase-1 from Alcanivorax borkumensis in Escherichia coli.

Enzymes with hydroxylating activity on alkanes have potential application as biotransformation catalysts in chemical and pharmaceutical industry. Genome of Alcanivorax borkumensis, a marine bacterium with hydrocarbon dissimilation activity, contains at least two P450 monooxygenases and two nonheme monooxygenases, AlkB1 and AlkB2, respectively. Presumably, all these enzymes possess alkane hydroxylating activity. Both AlkB1 and AlkB2 are membrane proteins. Two accessory proteins, rubredoxin and rubredoxin reductase, supply the reducing equivalent from nicotinamide adenine dinucleotide phosphate reduced (NADPH to hydroxylases. Rubredoxin reductase catalyses the reduction of rubredoxin by oxidation of NADPH, and rubredoxin transfers the electrons to the alkane hydroxylase to complete the hydroxylation reaction. Here, we sought to investigate the expression of alkB1 gene in Escherichia coli. Therefore, we amplified alkB1 gene from A. borkumensis genome by polymerase chain reaction and cloned it in the expression vector pET26 upstream of His-tag sequence. Predisposed BL21 (DE3) cells were transformed by the recombinant vector. At last, expression of recombinant enzyme was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Regarding the potential ability of this enzyme in hydroxylation of long-chained alkanes, the application of it would be studied in petroleum downstream industries.

Induction of apoptosis and CYP4A1 expression in Sprague-Dawley rats exposed to low doses of perfluorooctane sulfonate.

In previous studies, perfluorooctane sulfonate (PFOS), an environmental organic compound, was reported to cause hepatotoxicity and hypolipidemia in rodents. However, the low dose toxicity of PFOS and the toxic mechanisms involved remain to be determined. To clarify the low dose toxicity and action mechanism in the target organ toxicity, Sprague-Dawley (SD) rats were orally administered with PFOS at the doses of 0, 1.25, 5, 10 mg/kg/day for 28 days. As a result, no death or abnormal symptoms were observed in all groups. The significant loss of mean body weight was observed in female rats treated with 10 mg/kg PFOS and the relative liver weight of 10 mg/kg PFOS-treated group was significantly greater compared to control. Histopathological examination revealed that fatty change was evident in the liver of male rats treated with PFOS (5 and 10 mg/kg) and hypertrophy and cellular swellings in females at the dose of 10 mg/kg, which showed different pattern of pathological lesions. In addition, we demonstrated the expression induction of hepatic caspase-3 and cytochrome P450 4A1 (CYP4A1) related with apoptosis and lipid metabolism, respectively. This study suggested that no-observed-adverse-effect level (NOAEL) of PFOS was 1.25 mg/kg in 28-day repeated toxicity study and, however, the toxic response showed gender differences. The possible toxic mechanism of PFOS was the induction of apoptosis and altering lipid metabolism which resulted in hepatotoxicity.

Piroxicam reverses endotoxin-induced hypotension in rats: contribution of vasoactive eicosanoids and nitric oxide.

Nitric oxide (NO) produced by inducible NO synthase (iNOS) is responsible for endotoxin-induced vascular hyporeactivity and hypotension resulting in multiple organ failure. Endotoxic shock is also characterized by decreased expression of constitutive cyclooxygenase (COX-1), cytochrome P450 (CYP) 4A and endothelial NOS (eNOS). Our previous studies demonstrated that dual inhibition of iNOS and COX with a selective COX-2 inhibitor, NS-398, or a non-selective COX inhibitor, indomethacin, restores blood pressure presumably because of increased production of 20-hydroxyeicosatetraenoic acid (20-HETE) derived from arachidonic acid (AA) by CYP4A in endotoxaemic rats. The aim of this study was to investigate the effects of piroxicam, a preferential COX-1 inhibitor, on the endotoxin-induced changes in blood pressure, expression of COX-1, inducible COX (COX-2), CYP4A1, eNOS, iNOS and heat shock protein 90 (hsp90), and production of PGI(2), PGE(2), 20-HETE and NO. Injection of endotoxin (10 mg/kg, i.p.) to male Wistar rats caused a fall in blood pressure and an increase in heart rate associated with elevated renal 6-keto-PGF(1α) and PGE(2) levels as well as an increase in COX-2 protein expression. Endotoxin also caused an elevation in systemic and renal nitrite levels associated with increased renal iNOS protein expression. In contrast, systemic and renal 20-HETE levels and renal expression of eNOS, COX-1 and CYP4A1 were decreased in endotoxaemic rats. The effects of endotoxin, except for renal COX-1 and eNOS protein expression, were prevented by piroxicam (10 mg/kg, i.p.), given 1 hr after injection of endotoxin. Endotoxin did not change renal hsp90 protein expression. These data suggest that a decrease in the expression and activity of COX-2 and iNOS associated with an increase in CYP4A1 expression and 20-HETE synthesis contributes to the effect of piroxicam to prevent the hypotension during rat endotoxaemia.

Specific accumulation of CYP94A1 transcripts after exposure to gaseous benzaldehyde: induction of lauric acid ω-hydroxylase activity in Vicia sativa exposed to atmospheric pollutants.

The effects of air pollutants such as aldehydes, ozone, nitrogen dioxide and benzene on fatty acid ω-hydroxylase activity in Vicia sativa microsomes have been investigated. Four days old etiolated V. sativa seedlings were exposed to different concentrations of selected pollutants for varying exposure times. Growing etiolated V. sativa seedlings in air containing the gaseous benzaldehyde (150 nM) led to an 8-fold enhancement of lauric acid ω-hydroxylase activity in microsomes of treated plants compared to controls grown in pure air (96 ± 10 versus 12 ± 2 pmol/min/mg protein, respectively). The induction increased with increasing gas phase concentrations (10-1300 nM) and the maximum of activity was measured after 48 h of exposure. Northern blot analysis revealed that this induction occurred via transcriptional activation of the gene coding for CYP94A1. The absence of CYP94A2 and CYP94A3 transcription activation together with the missing effect on epoxide hydrolases activities indicate the specificity of CYP94A1 induction by benzaldehyde. Exposure to nitrogen dioxide, ozone and formaldehyde also stimulated lauric acid ω-hydroxylases activity while exposure to benzene did not show any effect.

Production of a recombinant alkane hydroxylase (AlkB2) from Alcanivorax borkumensis.

Alcanivorax borkumensis is an oil-degrading marine bacterium. Its genome contains genes coding for three cytochrome P450s and two integral membrane alkane hydroxylases (AlkB1 & AlkB2), all assumed to perform hydroxylation of different linear or branched alkanes. Although, the sequence of alkB2 has been determined, the molecular characterization and the substrate specificity of AlkB2 require more precise investigation. In this study, AlkB2 from A. borkumensis SK2 was expressed in Escherichia coli to examine the functionality of AlkB2 as a hydroxylating enzyme. Furthermore, the activity of the enzyme in the presence of the accessory proteins, rubredoxin (RubA) and rubredoxin reductase (RubB), produced in E. coli BL21(DE3)plysS cells, was determined. Recombinant AlkB2 is produced in an active form and rubredoxin is the intermediate electron donor to AlkB2 and can replace AlkG function, when NADH is the prime electron donor.

Expression and characterization of CYP4V2 as a fatty acid omega-hydroxylase.

Bietti's crystalline dystrophy is an ocular disease that is strongly associated with polymorphisms in the CYP4V2 gene. CYP4 enzymes are typically microsomal fatty acid omega-hydroxylases that function together with mitochondrial and peroxisomal beta-oxidation enzymes to degrade cellular lipids. Indeed, ocular and peripheral cells cultured from patients with Bietti's have been reported to exhibit abnormal lipid metabolism. However, CYP4V2 possesses low sequence homology to other members of the CYP4 family. Therefore, we cloned and expressed CYP4V2 and analyzed the functional characteristics of this new cytochrome P450 enzyme. We find that CYP4V2 is a selective omega-hydroxylase of saturated, medium-chain fatty acids with relatively high catalytic efficiency toward myristic acid. Moreover, N-hydroxy-N'-(4-n-butyl-2-methylphenyl formamidine) (HET0016) is a nanomolar inhibitor of the enzyme. Therefore, CYP4V2 exhibits catalytic functions typical of a human CYP4 enzyme, but with a distinctive chain-length selectivity coupled with high omega-hydroxylase specificity. Consequently, defective omega-oxidation of ocular fatty acids/lipids secondary to mutations in the CYP4V2 gene appears to be a plausible mechanism underlying Bietti's crystalline dystrophy.

Administration of ciprofibrate to lactating mothers induces PPARalpha-signaling pathway in the liver and kidney of suckling rats.

It is well known that the hypolipidemic drug ciprofibrate induces peroxisome proliferation in rodent liver, which in turn leads to the oxidative stress, and modifies some parameters related to cell proliferation and apoptosis. The administration of ciprofibrate to rats during the lactating period determined in their pups significant modifications in hepatic peroxisome enzyme activities, induction of the PPARalpha-target gene, Cyp4a10, and perturbation in cell proliferation and apoptosis, which affected the size of the liver. Moreover, this modification was associated to about two-fold induction of mRNA-PPARalpha. On the contrary, in the kidney, although a similar two-fold up-regulation of PPARalpha was detected, the induction of both peroxisomal enzyme activities and Cyp4a10 were weak, and no alterations were detected, neither in cell cycle nor in the size of the tissue. Our results indicate that the response to ciprofibrate is stronger in the liver than in the kidney of newborn rats.

Deficient renal 20-HETE release in the diabetic rat is not the result of oxidative stress.

We confirmed that release of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated perfused kidney of diabetic rats is greatly reduced compared with age-matched control rats. The present studies were undertaken to examine potential mechanisms for the deficit in renal 20-HETE in rats with streptozotocin-induced diabetes of 3-4 wk duration. A role for oxidative stress was excluded, inasmuch as treatment of diabetic rats with tempol, an SOD mimetic, for 4 wk did not affect the renal release of 20-HETE. Similarly, chronic inhibition of nitric oxide formation with nitro-l-arginine methyl ester or aldose reductase with zopolrestat failed to alter the release of 20-HETE from the diabetic rat kidney. Inasmuch as 20-HETE may be metabolized by cyclooxygenase (COX), the expression/activity of which is increased in diabetes, we included indomethacin in the perfusate of the isolated kidney to inhibit COX but found no effect on 20-HETE release. Diabetic rats were treated for 3 wk with fenofibrate to increase expression of cytochrome P-450 (CYP4A) in an attempt to find an intervention that would restore release of 20-HETE from the diabetic rat kidney. However, fenofibrate reduced 20-HETE release in diabetic and control rat kidneys but increased expression of CYP4A. Only insulin treatment of diabetic rats for 2 wk to reverse the hyperglycemia and maintain blood glucose levels at <200 mg/dl reversed the renal deficit in 20-HETE. We conclude that oxidative stress, increased aldose reductase activity, or increased COX activity does not contribute to the renal deficit of 20-HETE in diabetes, which may be directly related to insulin deficiency.

Evidence for involvement of copper ions and redox state in regulation of butane monooxygenase in Pseudomonas butanovora.

Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C(2)-C(9) alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of beta-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O(2) when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH(4)Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O(2) (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O(2), CuSO(4) (0.5 microM) repressed 1-butanol-dependent induction of beta-galactosidase activity. Under oxic conditions (20% O(2) [vol/vol]), significantly higher concentrations of CuSO(4) (2 microM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu(2+) reducing agent Na ascorbate (100 microM) and CuSO(4) (0.5 microM) repressed the induction of beta-galactosidase activity under oxic conditions to the same extent that 0.5 microM CuSO(4) alone repressed it under anoxic conditions. Under oxic conditions, 2 microM CuSO(4) repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO(4) repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

A new model for nonalcoholic steatohepatitis in the rat utilizing total enteral nutrition to overfeed a high-polyunsaturated fat diet.

We have used total enteral nutrition (TEN) to moderately overfeed rats high-polyunsaturated fat diets to develop a model for nonalcoholic steatohepatitis (NASH). Male Sprague-Dawley rats were fed by TEN a 187 kcal.kg(-3/4).day(-1) diet containing 5% (total calories) corn oil or a 220 kcal.kg(-3/4).day(-1) diet in which corn oil constituted 5, 10, 25, 35, 40, or 70% of total calories for 21 or 65 days. Rats fed the 5% corn oil, 220 kcal.kg(-3/4).day(-1)diet had greater body weight gain (P < or = 0.05), fat mass (P < or = 0.05), and serum leptin and glucose levels (P < or = 0.05), but no liver pathology. A dose-dependent increase in hepatic triglyceride deposition occurred with increase in percent corn oil in the 220 kcal.kg(-3/4).day(-1) groups (P < or = 0.05). Steatosis, macrophage infiltration, apoptosis, and focal necrosis were present in the 70% corn oil group, accompanied by elevated serum alanine aminotransferase (ALT) levels (P < or = 0.05). An increase in oxidative stress (thiobarbituric acid-reactive substances) and TNF-alpha expression (P < or = 0.05) was observed in the 70% corn oil group, as well as an increase in hepatic CYP2E1 and CYP4A1 expression (P < or = 0.05). Significant positive correlations were observed between the level of dietary corn oil and the degree of pathology, ALTs, oxidative stress, and inflammation. Liver pathology was progressive with increased necrosis, accompanied by fibrosis, observed after 65 days of TEN. Increased expression of CD36 and l-fabp mRNA suggested development of steatosis was associated with increased fatty acid transport. These data suggest that intragastric infusion of a high-polyunsaturated fat diet at a caloric level of 17% excess total calories results in pathology similar to clinical NASH.