Dietary alteration of n-3 and n-6 fatty acids for headache reduction in adults with migraine: randomized controlled trial.

OBJECTIVE: To determine whether dietary interventions that increase n-3 fatty acids with and without reduction in n-6 linoleic acid can alter circulating lipid mediators implicated in headache pathogenesis, and decrease headache in adults with migraine. DESIGN: Three arm, parallel group, randomized, modified double blind, controlled trial. SETTING: Ambulatory, academic medical center in the United States over 16 weeks. PARTICIPANTS: 182 participants (88% women, mean age 38 years) with migraines on 5-20 days per month (67% met criteria for chronic migraine). INTERVENTIONS: Three diets designed with eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and linoleic acid altered as controlled variables: H3 diet (n=61)-increase EPA+DHA to 1.5 g/day and maintain linoleic acid at around 7% of energy; H3-L6 diet (n=61)-increase n-3 EPA+DHA to 1.5 g/day and decrease linoleic acid to ≤1.8% of energy; control diet (n=60)-maintain EPA+DHA at <150 mg/day and linoleic acid at around 7% of energy. All participants received foods accounting for two thirds of daily food energy and continued usual care. MAIN OUTCOME MEASURES: The primary endpoints (week 16) were the antinociceptive mediator 17-hydroxydocosahexaenoic acid (17-HDHA) in blood and the headache impact test (HIT-6), a six item questionnaire assessing headache impact on quality of life. Headache frequency was assessed daily with an electronic diary. RESULTS: In intention-to-treat analyses (n=182), the H3-L6 and H3 diets increased circulating 17-HDHA (log ng/mL) compared with the control diet (baseline-adjusted mean difference 0.6, 95% confidence interval 0.2 to 0.9; 0.7, 0.4 to 1.1, respectively). The observed improvement in HIT-6 scores in the H3-L6 and H3 groups was not statistically significant (-1.6, -4.2 to 1.0, and -1.5, -4.2 to 1.2, respectively). Compared with the control diet, the H3-L6 and H3 diets decreased total headache hours per day (-1.7, -2.5 to -0.9, and -1.3, -2.1 to -0.5, respectively), moderate to severe headache hours per day (-0.8, -1.2 to -0.4, and -0.7, -1.1 to -0.3, respectively), and headache days per month (-4.0, -5.2 to -2.7, and -2.0, -3.3 to -0.7, respectively). The H3-L6 diet decreased headache days per month more than the H3 diet (-2.0, -3.2 to -0.8), suggesting additional benefit from lowering dietary linoleic acid. The H3-L6 and H3 diets altered n-3 and n-6 fatty acids and several of their nociceptive oxylipin derivatives in plasma, serum, erythrocytes or immune cells, but did not alter classic headache mediators calcitonin gene related peptide and prostaglandin E2. CONCLUSIONS: The H3-L6 and H3 interventions altered bioactive mediators implicated in headache pathogenesis and decreased frequency and severity of headaches, but did not significantly improve quality of life. TRIAL REGISTRATION: ClinicalTrials.gov NCT02012790.

Molecular Pathways Linking Oxylipins to Nociception in Rats.

Oxylipins are lipid peroxidation products that participate in nociceptive, inflammatory, and vascular responses to injury. Effects of oxylipins depend on tissue-specific differences in accumulation of precursor polyunsaturated fatty acids and the expression of specific enzymes to transform the precursors. The study of oxylipins in nociception has presented technical challenges leading to critical knowledge gaps in the way these molecules operate in nociception. We applied a systems-based approach to characterize oxylipin precursor fatty acids, and expression of genes coding for proteins involved in biosynthesis, transport, signaling and inactivation of pro- and antinociceptive oxylipins in pain circuit tissues. We further linked these pathways to nociception by demonstrating intraplantar carrageenan injection induced gene expression changes in oxylipin biosynthetic pathways. We determined functional-biochemical relevance of the proposed pathways in rat hind paw and dorsal spinal cord by measuring basal and stimulated levels of oxylipins throughout the time-course of carrageenan-induced inflammation. Finally, when oxylipins were administered by intradermal injection we observed modulation of nociceptive thermal hypersensitivity, providing a functional-behavioral link between oxylipins, their molecular biosynthetic pathways, and involvement in pain and nociception. Together, these findings advance our understanding of molecular lipidomic systems linking oxylipins and their precursors to nociceptive and inflammatory signaling pathways in rats. PERSPECTIVE: We applied a systems approach to characterize molecular pathways linking precursor lipids and oxylipins to nociceptive signaling. This systematic, quantitative evaluation of the molecular pathways linking oxylipins to nociception provides a framework for future basic and clinical research investigating the role of oxylipins in pain.

Identifying oxidized lipid mediators as prognostic biomarkers of chronic posttraumatic headache.

Chronic posttraumatic headache (PTH) is among the most common and disabling sequelae of traumatic brain injury (TBI). Current PTH treatments are often only partially effective and have problematic side effects. We previously showed in a small randomized trial of patients with chronic nontraumatic headaches that manipulation of dietary fatty acids decreased headache frequency, severity, and pain medication use. Pain reduction was associated with alterations in oxylipins derived from n-3 and n-6 fatty acids, suggesting that oxylipins could potentially mediate clinical pain reduction. The objective of this study was to investigate whether circulating oxylipins measured in the acute setting after TBI could serve as prognostic biomarkers for developing chronic PTH. Participants enrolled in the Traumatic Head Injury Neuroimaging Classification Protocol provided serum within 3 days of TBI and were followed up at 90 days postinjury with a neurobehavioral symptom inventory (NSI) and satisfaction with life survey. Liquid chromatography-tandem mass spectrometry methods profiled 39 oxylipins derived from n-3 docosahexaenoic acid (DHA), and n-6 arachidonic acid and linoleic acid. Statistical analyses assessed the association of oxylipins with headache severity (primary outcome, measured by headache question on NSI) as well as associations between oxylipins and total NSI or satisfaction with life survey scores. Among oxylipins, 4-hydroxy-DHA and 19,20-epoxy-docosapentaenoate (DHA derivatives) were inversely associated with headache severity, and 11-hydroxy-9-epoxy-octadecenoate (a linoleic acid derivative) was positively associated with headache severity. These findings support a potential for DHA-derived oxylipins as prognostic biomarkers for development of chronic PTH.

Plasma oxylipins and unesterified precursor fatty acids are altered by DHA supplementation in pregnancy: Can they help predict risk of preterm birth?

Oxidized lipids derived from omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids, collectively known as oxylipins, are bioactive signaling molecules that play diverse roles in human health and disease. Supplementation with n-3 docosahexaenoic acid (DHA) during pregnancy has been reported to decrease the risk of preterm birth in singleton pregnancies, which may be due to effects of DHA supplementation on oxylipins or their precursor n-6 and n-3 fatty acids. There is only limited understanding of the levels and trajectory of changes in plasma oxylipins during pregnancy, effects of DHA supplementation on oxylipins and unesterified fatty acids, and whether and how oxylipins and their unesterified precursor fatty acids influence preterm birth. In the present study we used liquid chromatography-tandem mass spectrometry to profile oxylipins and their precursor fatty acids in the unesterified pool using plasma samples collected from a subset of pregnant Australian women who participated in the ORIP (Omega-3 fats to Reduce the Incidence of Prematurity) study. ORIP is a large randomized controlled trial testing whether daily supplementation with n-3 DHA can reduce the incidence of early preterm birth compared to control. Plasma was collected at study entry (≈pregnancy week 14) and again at ≈week 24, in a subgroup of 48 ORIP participants-12 cases with spontaneous preterm (<37 weeks) birth and 36 matched controls with spontaneous term (≥40 weeks) birth. In the combined preterm and term pregnancies, we observed that in the control group without DHA supplementation unesterified AA and AA-derived oxylipins 12-HETE, 15-HETE and TXB2 declined between weeks 14-24 of pregnancy. Compared to control, DHA supplementation increased unesterified DHA, EPA, and AA, DHA-derived 4-HDHA, 10-HDHA and 19,20-EpDPA, and AA-derived 12-HETE at 24 weeks. In exploratory analysis independent of DHA supplementation, participants with concentrations above the median for 5-lipoxygenase derivatives of AA (5-HETE, Odds Ratio (OR) 8.2; p = 0.014) or DHA (4-HDHA, OR 8.0; p = 0.015) at 14 weeks, or unesterified AA (OR 5.1; p = 0.038) at 24 weeks had higher risk of spontaneous preterm birth. The hypothesis that 5-lipoxygenase-derived oxylipins and unesterified AA could serve as mechanism-based biomarkers predicting spontaneous preterm birth should be evaluated in larger, adequately powered studies.

Temperature and time-dependent effects of delayed blood processing on oxylipin concentrations in human plasma.

BACKGROUND: Oxidized derivatives of polyunsaturated fatty acids, collectively known as oxylipins, are labile bioactive mediators with diverse roles in human physiology and pathology. Oxylipins are increasingly being measured in plasma collected in clinical studies to investigate biological mechanisms and as pharmacodynamic biomarkers for nutrient-based and drug-based interventions. Whole blood is generally stored either on ice or at room temperature prior to processing. However, the potential impacts of delays in processing, and of temperature prior to processing, on oxylipin concentrations are incompletely understood. OBJECTIVE: To evaluate the effects of delayed processing of blood samples in a timeframe that is typical of a clinical laboratory setting, using typical storage temperatures, on concentrations of representative unesterified oxylipins measured by liquid chromatography-tandem mass spectrometry. DESIGN: Whole blood (drawn on three separate occasions from a single person) was collected into 5 mL purple-top potassium-EDTA tubes and stored for 0, 10, 20, 30, 60 or 120 min at room temperature or on wet ice, followed by centrifugation at 4 °C for 10 min with plasma collection. Each sample was run in duplicate, therefore there were six tubes and up to six data points at each time point for each oxylipin at each condition (ice/room temperature). Representative oxylipins derived from arachidonic acid, docosahexaenoic acid, and linoleic acid were quantified by liquid chromatography tandem mass spectrometry. Longitudinal models were used to estimate differences between temperature groups 2 h after blood draw. RESULTS: We found that most oxylipins measured in human plasma in traditional potassium-EDTA tubes are reasonably stable when stored on ice for up to 2 h prior to processing, with little evidence of auto-oxidation in either condition. By contrast, in whole blood stored at room temperature, substantial time-dependent increases in the 12-lipoxygenase-derived (12-HETE, 14-HDHA) and platelet-derived (thromboxane B2) oxylipins were observed. CONCLUSION: These findings suggest that certain plasma oxylipins can be measured with reasonable accuracy despite delayed processing for up to 2 h when blood is stored on ice prior to centrifugation. 12-Lipoxygenase- and platelet-derived oxylipins may be particularly sensitive to post-collection artifact with delayed processing at room temperature. Future studies are needed to determine impacts of duration and temperature of centrifugation on oxylipin concentrations.

Lipidomic profiling of targeted oxylipins with ultra-performance liquid chromatography-tandem mass spectrometry.

Oxylipins are bioactive mediators that play diverse roles in (patho)physiology. We developed a sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous profiling of 57 targeted oxylipins derived from five major n-6 and n-3 polyunsaturated fatty acids (PUFAs) that serve as oxylipin precursors, including linoleic (LA), arachidonic (AA), alpha-linolenic (ALA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids. The targeted oxylipin panel provides broad coverage of lipid mediators and pathway markers generated from cyclooxygenases, lipoxygenases, cytochrome P450 epoxygenases/hydroxylases, and non-enzymatic oxidation pathways. The method is based on combination of protein precipitation and solid-phase extraction (SPE) for sample preparation, followed by UPLC-MS/MS. This is the first methodology to incorporate four hydroxy-epoxy-octadecenoic acids and four keto-epoxy-octadecenoic acids into an oxylipin profiling network. The novel method achieves excellent resolution and allows in-depth analysis of isomeric and isobaric species of oxylipin extracts in biological samples. The method was quantitatively characterized in human plasma with good linearity (R = 0.990-0.999), acceptable reproducibility (relative standard deviation (RSD) < 20% for the majority of analytes), accuracy (67.8 to 129.3%) for all analytes, and recovery (66.8-121.2%) for all analytes except 5,6-EET. Ion enhancement effects for 28% of the analytes in tested concentrations were observed in plasma, but were reproducible with RSD < 17.2%. Basal levels of targeted oxylipins determined in plasma and serum are in agreement with those previously reported in literature. The method has been successfully applied in clinical and preclinical studies.

Bioactive Lipid Mediator Profiles in Human Psoriasis Skin and Blood.

Psoriasis is a chronic immune-mediated disease that represents a unique model for investigating inflammation at local and systemic levels. Bioactive lipid mediators (LMs) are potent compounds reported to play a role in the development and resolution of inflammation. Currently, it is not known to what extent these LMs are involved in psoriasis pathophysiology and related metabolic dysfunction. Here, we use targeted and untargeted liquid chromatography-tandem mass spectrometry approaches to quantify LMs in skin and peripheral blood from psoriasis patients and compared them with those of healthy individuals. Lesional psoriasis skin was abundant in arachidonic acid metabolites, as 8-, 12- and 15-hydroxyeicosatetraenoic acid, compared with adjacent nonlesional and skin from healthy individuals. Additionally, a linoleic acid-derived LM, 13-hydroxyoctadecadienoic acid, was significantly increased compared with healthy skin (607.9 ng/g vs. 5.4 ng/g, P = 0.001). These psoriasis skin differences were accompanied by plasma decreases in antioxidant markers, including glutathione, and impaired lipolysis characterized by lower concentrations of primary and secondary bile acids. In conclusion, our study shows that psoriasis skin and blood have disease-specific phenotype profiles of bioactive LMs represented by omega-6 fatty acid-oxidized derivatives. These findings provide insights into psoriasis pathophysiology that could potentially contribute to new biomarkers and therapeutics.

A systems approach for discovering linoleic acid derivatives that potentially mediate pain and itch.

Chronic pain and itch are common hypersensitivity syndromes that are affected by endogenous mediators. We applied a systems-based, translational approach to predict, discover, and characterize mediators of pain and itch that are regulated by diet and inflammation. Profiling of tissue-specific precursor abundance and biosynthetic gene expression predicted that inflamed skin would be abundant in four previously unknown 11-hydroxy-epoxy- or 11-keto-epoxy-octadecenoate linoleic acid derivatives and four previously identified 9- or 13-hydroxy-epoxy- or 9- or 13-keto-epoxy-octadecenoate linoleic acid derivatives. All of these mediators were confirmed to be abundant in rat and human skin by mass spectrometry. However, only the two 11-hydroxy-epoxy-octadecenoates sensitized rat dorsal root ganglion neurons to release more calcitonin gene-related peptide (CGRP), which is involved in pain transmission, in response to low pH (which mimics an inflammatory state) or capsaicin (which activates ion channels involved in nociception). The two 11-hydroxy-epoxy-octadecenoates share a 3-hydroxy-Z-pentenyl-E-epoxide moiety, thus suggesting that this substructure could mediate nociceptor sensitization. In rats, intradermal hind paw injection of 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate elicited C-fiber-mediated sensitivity to thermal pain. In a randomized trial testing adjunctive strategies to manage refractory chronic headaches, reducing the dietary intake of linoleic acid was associated with decreases in plasma 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate, which correlated with clinical pain reduction. Human psoriatic skin had 30-fold higher 9-keto-12,13-trans-epoxy-(10E)-octadecenoate compared to control skin, and intradermal injection of this compound induced itch-related scratching behavior in mice. Collectively, these findings define a family of endogenous mediators with potential roles in pain and itch.

Transient postnatal fluoxetine decreases brain concentrations of 20-HETE and 15-epi-LXA4, arachidonic acid metabolites in adult mice.

BACKGROUND: Transient postnatal exposure of rodents to the selective serotonin (5-HT) reuptake inhibitor (SSRI) fluoxetine alters behavior and brain 5-HT neurotransmission during adulthood, and also reduces brain arachidonic (ARA) metabolic consumption and protein level of the ARA metabolizing enzyme, cytochrome P4504A (CYP4A). HYPOTHESIS: Brain 20-hydroxyeicosatetraenoic acid (20-HETE), converted by CYP4A from ARA, will be reduced in adult mice treated transiently and postnatally with fluoxetine. METHODS: Male mice pups were injected i.p. daily with fluoxetine (10mg/kg) or saline during P4-P21. At P90 their brain was high-energy microwaved and analyzed for 20-HETE and six other ARA metabolites by enzyme immunoassay. RESULTS: Postnatal fluoxetine vs. saline significantly decreased brain concentrations of 20-HETE (-70.3%) and 15-epi-lipoxin A4 (-60%) in adult mice, but did not change other eicosanoid concentrations. CONCLUSIONS: Behavioral changes in adult mice treated postnatally with fluoxetine may be related to reduced brain ARA metabolism involving CYP4A and 20-HETE formation.

Low-dose aspirin (acetylsalicylate) prevents increases in brain PGE2, 15-epi-lipoxin A4 and 8-isoprostane concentrations in 9 month-old HIV-1 transgenic rats, a model for HIV-1 associated neurocognitive disorders.

BACKGROUND: Older human immunodeficiency virus (HIV)-1 transgenic rats are a model for HIV-1 associated neurocognitive disorders (HAND). They show behavioral changes, neuroinflammation, neuronal loss, and increased brain arachidonic acid (AA) enzymes. Aspirin (acetylsalicylate, ASA) inhibits AA oxidation by cyclooxygenase (COX)-1 and COX-2. HYPOTHESIS: Chronic low-dose ASA will downregulate brain AA metabolism in HIV-1 transgenic rats. METHODS: Nine month-old HIV-1 transgenic and wildtype rats were given 42 days of 10mg/kg/day ASA or nothing in drinking water; eicosanoids were measured using ELISAs on microwaved brain extracts. RESULTS: Brain 15-epi-lipoxin A4 and 8-isoprostane concentrations were significantly higher in HIV-1 transgenic than wildtype rats; these differences were prevented by ASA. ASA reduced prostaglandin E2 and leukotriene B4 concentrations in HIV-1 Tg but not wildtype rats. Thromboxane B2, 15-HETE, lipoxin A4 and resolvin D1 concentrations were unaffected by genotype or treatment. CONCLUSION: Chronic low-dose ASA reduces AA-metabolite markers of neuroinflammation and oxidative stress in a rat model for HAND.

Identification and profiling of targeted oxidized linoleic acid metabolites in rat plasma by quadrupole time-of-flight mass spectrometry.

Linoleic acid (LA) and LA-esters are the precursors of LA hydroperoxides, which are readily converted to 9- and 13-hydroxy-​octadecadienoic acid (HODE) and 9- and 13-oxo-​octadecadienoic acid (oxo ODE) metabolites in vivo. These four oxidized LA metabolites (OXLAMs) have been implicated in a variety of pathological conditions. Therefore, their accurate measurement may provide mechanistic insights into disease pathogenesis. Here we present a novel quadrupole time-of-flight mass spectrometry (Q-TOFMS) method for quantitation and identification of target OXLAMs in rat plasma. In this method, the esterified OXLAMs were base-hydrolyzed and followed by liquid-liquid extraction. Quantitative analyses were based on one-point standard addition with isotope dilution. The Q-TOFMS data of target metabolites were acquired and multiple reaction monitoring extracted-ion chromatograms were generated post-acquisition with a 10 ppm extraction window. The limit of quantitation was 9.7-35.9 nmol/L depending on the metabolite. The method was reproducible with a coefficient of variation of <18.5%. Mean concentrations of target metabolites in rat plasma were 57.8, 123.2, 218.1 and 57.8 nmol/L for 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE, respectively. Plasma levels of total OXLAMs were 456.9 nmol/L, which correlated well with published concentrations obtained by gas chromatography/mass spectrometry (GC/MS). The concentrations were also obtained utilizing a standard addition curve approach. The calibration curves were linear with correlation coefficients of >0.991. Concentrations of 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE were 84.0, 138.6, 263.0 and 69.5 nmol/L, respectively, which were consistent with the results obtained from one-point standard addition. Target metabolites were simultaneously characterized based on the accurate Q-TOFMS data. This is the first study of secondary LA metabolites using Q-TOFMS. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Metabolites of the carcinogen 2-amino-alpha-carboline formed in male Sprague-Dawley rats in vivo and in rat hepatocyte and human HepG2 cell incubates.

2-amino-alpha-carboline (AaC, 2-amino-9H-pyrido[2,3-b]indole) is a genotoxic carcinogen produced by cooking of protein-containing foods and combustion of biomaterial. Humans are chronically exposed to low levels of AaC through foods (grilled or pan-fried meats), drinking water, and smoke inhalation (cigarette/wood smoke, diesel exhaust). We report herein 17 metabolites of AaC formed in vivo in male Sprague-Dawley rats (from bile, urine, and plasma) and in situ in rat hepatocytes and human HepG2 liver tumor cells. We confirmed several expected sites of AaC metabolism, but also observed novel metabolites. The novel metabolites include extensive N-acetylated AaC conjugates, multiple N-glucuronides, and at least one additional site of aromatic ring hydroxylation. The abundance of N-acetylated metabolites is noteworthy because this metabolic pathway is generally unrecognized for HAAs. Also noteworthy are metabolites that were not detected, i.e., no direct AaC N-sulfonation to form the sulfamate. These results, combined with earlier publications on the reactive (DNA adduct forming) metabolites of AaC, indicate that both bioactivation and detoxification of AaC share the same metabolic pathways--namely, oxidation, acetylation, and sulfonation. This may be an important factor attenuating the risk of carcinogenesis from AaC exposure; increased potential for bioactivation could be balanced by increased potential for detoxification.

Comparative metabolism of the aza polynuclear aromatic hydrocarbon dibenz[a,h]acridine by recombinant human and rat cytochrome P450s.

To assess the role of human and rat cytochrome P450s in the metabolism of aza-polynuclear aromatic hydrocarbons (aza-PAHs) and to examine the influence of heterocyclic nitrogen on the metabolism of these chemicals, we have investigated the biotransformation of dibenz[a,h]acridine (DB[a,h]ACR), an aza-PAH with two nonidentical bay regions, by recombinant human cytochromes P450 1A1, 1B1, and 3A4 and rat P450 1A1. Among the three P450s, 1A1 was the most effective in metabolizing DB[a,h]ACR followed by 1B1 and 3A4. The major DB[a,h]ACR metabolites produced by human P450 1A1 and 1B1 were the dihydrodiols with a bay region double bond, namely, DB[a,h]ACR-3,4-diol and DB[a,h]ACR-10,11-diol (putative proximate carcinogen). P450 1A1 produced a higher proportion of DB[a,h]ACR-10,11-diol (derived from the benzo ring adjacent to the nitrogen) (44.7%) than of DB[a,h]ACR-3,4-diol (derived from benzo ring away from the nitrogen) (23.8%). In contrast, 1B1 produced a much greater proportion of 3,4-diol (54.7%) than of 10,11-diol (6.4%). These data indicate that (i) human P450 1A1 and 1B1 differ dramatically with respect to the regiospecific metabolism of DB[a,h]ACR, (ii) human P450 1A1 is substantially more active than human P450 1B1 in the metabolic activation of the aza-PAH to its 10,11-diol, and (iii) the presence of nitrogen influences the relative extent to which the two benzo ring diols with a bay region double bond are formed by human P450s 1A1 and 1B1. In contrast to human P450s 1A1 and 1B1, rat P450 1A1 showed no regioselectivity in the metabolism of DB[a,h]ACR producing nearly equal proportions of 10,11-diol and 3,4-diol. Despite significant differences in their regioselectivity, human P450 1A1 and 1B1 and rat P450 1A1 showed similar stereoselectivity in the metabolism of DB[a,h]ACR to its diols having a bay region double bond, producing primarily the R,R enantiomers (>94%). The data of these studies indicate that human and rat P450 1A1 differ in their regioselectivity in the metabolism of DB[a,h]ACR to its two benzo ring diols with a bay region double bond and consequently in their ability to metabolically activate the parent aza-PAH. However, human and rat P450 1A1 do not differ with respect to their stereoselectivity in the metabolism of DB[a,h]ACR to the diols.

Metabolism of the polynuclear sulfur heterocycle benzo[b]phenanthro[2,3-d]thiophene by rodent liver microsomes: evidence for multiple pathways in the bioactivation of benzo[b]phenanthro[2,3-d]thiophene.

Benzo[b]phenanthro[2,3-d]thiophene (BPT), a thia analogue of dibenz[a,h]anthracene (DBA), is a carcinogenic environmental pollutant. We have examined the metabolism of BPT by rodent liver microsomes to investigate the mechanism by which BPT produces mutagenic and carcinogenic effects. Both rat and mouse liver microsomes biotransformed [G-(3)H]BPT to various metabolites including BPT 3,4-diol and BPT sulfoxide, which are significantly more mutagenic than the parent compound. Liver microsomes from both control mice and rats metabolize BPT at similar rates. Treatment of mice with P450 inducers DBA, 3-methylcholanthrene (3-MC), Aroclor 1254, and phenobarbital enhanced the rate of metabolism of BPT by 74-, 28-, 77-, and 6-fold, respectively. In comparison, the treatment of rats with DBA and 3-MC increased the rate of metabolism of BPT by 22- and 34-fold, respectively, suggesting that P450 enzymes responsible for the metabolism of BPT are enhanced to different extents in rats and mice by a similar class of compounds. In general, the liver microsomes from mice treated with DBA or 3-MC were more active than those from similarly treated rats in metabolizing BPT to its 3,4-diol, a precursor to the bay-region diol epoxide of BPT. BPT sulfone was a minor metabolite (if formed) in all cases. The liver microsomes from rats treated with DBA or 3-MC or from mice treated with PB produced a significant proportion of BPT sulfoxide (12-41%). In contrast, the liver microsomes from DBA- or 3-MC-treated mice formed BPT sulfoxide as a minor metabolite (<2%). These studies indicate that cytochrome P450 enzymes induced by PAHs (e.g., P450 1A1 and P450 1B1) and by PB (e.g., P450 2B1, 3A1, and/or 3A2) are involved in the metabolism of BPT to mutagenic BPT 3,4-diol and BPT sulfoxide, providing evidence for the first time that BPT and possibly other thia-PAHs are metabolically activated via the formation of both the dihydrodiol (and subsequently diol epoxide) and the sulfoxide.