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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.

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.

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.

Chronic pain is both a global public health concern and a serious source of personal suffering for which current treatments have limited efficacy. Recently, oxylipins derived from linoleic acid (LA), the most abundantly consumed polyunsaturated fatty acid in the modern diet, have been implicated as mediators of pain in the periphery and spinal cord. However, oxidized linoleic acid derived mediators (OXLAMs) remain understudied in the brain, particularly during pain states. In this study, we employed a mouse model of chronic inflammatory pain followed by a targeted lipidomic analysis of the animals’ amygdala and periaqueductal grey (PAG) using LC-MS/MS to investigate the effect of chronic inflammatory pain on oxylipin concentrations in these two brain nuclei known to participate in pain sensation and perception. From punch biopsies of these brain nuclei, we detected twelve OXLAMs in both the PAG and amygdala and one arachidonic acid derived mediator, 15-HETE, in the amygdala only. In the amygdala, we observed an overall decrease in the concentration of the majority of OXLAMs detected, while in the PAG the concentrations of only the epoxide LA derived mediators, 9,10-EpOME and 12,13-EpOME, and one trihydroxy LA derived mediator, 9,10,11-TriHOME, were reduced. This data provides the first evidence that OXLAM concentrations in the brain are affected by chronic pain, suggesting that OXLAMs may be relevant to pain signaling and adaptation to chronic pain in pain circuits in the brain and that the current view of OXLAMs in nociception derived from studies in the periphery is incomplete.

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.

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 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

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 10 mg/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.

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-(9 Z )-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-(9 Z )-octadecenoate, which correlated with clinical pain reduction. Human psoriatic skin had 30-fold higher 9-keto-12,13- trans -epoxy-(10 E )-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.

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 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.

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.

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.

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.

The ability of Sphingomonas paucimobilis strain EPA 505 (a soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth) to metabolize 3-methyl-methylfluoranthene, 7-methlybenz[ a ]anthracene, 5-methylchrysene, and their corresponding unsubstituted parent hydrocarbons was investigated. The rate of degradation of 3-methylfluoranthene, 7-methylbenz[ a ]anthracene, and 5-methylchrysene by a resting cell suspension of S. paucimobilis grown on fluoranthene was 20.4, 6.2, and 2.7 nmole/mg of wet cells/hr, respectively. The rate of degradation of fluoranthene, benz[ a ]anthracene, and chrysene was 10.7, 3.4, and 1.4 nmole/mg of wet cells/hr, indicating that methylated PAHs are degraded at a higher rate than the corresponding unsubstituted analogs. Two major isolated metabolites of benz[ a ]anthracene were identified as 2-hydroxy-3-naphthoic acid, and 2-hydroxy-3-phenanthroic acid. Unlike benz[ a ]anthracene, 7-methylbenz[ a ]anthracene was metabolized to its cis -1,2-diol t...

3-Nitrobenzanthrone (NBA) is a suspected human carcinogen and has been identified in diesel exhaust and in airborne particulates. Human exposure to NBA is thought to occur primarily via the respiratory tract and bronchial as well as alveolar epithelial cells are believed to be primary targets for lung carcinogenesis. Nitroarenes require metabolic activation to DNA binding metabolites to become genotoxic carcinogens. In this study the metabolism of NBA as well as its metabolic intermediate 3-nitrosobenzanthrone was investigated in cultures of rat lung alveolar type II cells, in human fetal bronchial (HFBE) and rat bronchial epithelial (R3/1) as well as mesenchymal Rwd009 cells. 3-Aminobenzanthrone (ABA) was identified as the major metabolite from both substrates, but also small amounts of 3-acetyl-ABA were observed during short term incubations (6 to 24 h) with NBA. Inhibition studies with allopurinol in alveolar type II cells indicate that the cytosolic enzyme xanthine oxidase contributes substan...

In order to assess the differences in the ability of fish and rat liver to metabolize carcinogenic polycyclic aromatic hydrocarbons (PAHs), we have investigated the metabolism of dibenzo[a,l]pyrene (DB[a,l]P), a highly potent carcinogenic PAH, by liver microsomes from 3-methylcholanthrene-treated Shasta rainbow trout ( Oncorhynchus mykiss ) and rats. Rat liver microsomes metabolized DB[a,l]P at a slightly higher rate (1.3-fold) than trout liver microsomes. Compared to benzo[a]pyrene (B[a]P), DB[a,l]P was metabolized at a significantly lower rate by both rat and trout liver microsomes. Although the microsomes from the two species metabolized DB[a,l]P to qualitatively similar metabolites, they showed significant differences in the profile of the metabolites formed. The proportion of DB[a,l]P-11,12-diol, the proximate carcinogen of DB[a,l]P, formed by trout microsomes was over two-fold greater (32.6%) than the corresponding value for rat microsomes (15.6%). Unlike rat microsomes, trout microsomes metabolized DB[a,l]P to its K-region diol (8,9-diol) to a small extent (26.1 vs 3.6%). As previously noted with B[a]P, trout liver, compared to rat liver, appears to be more efficient in forming the proximate carcinogenic metabolite of DB[a,l]P but less efficient in producing its K-region diol, a non-carcinogenic metabolite.

Tamoxifen has been shown to be a potent liver carcinogen in rats, and generates covalent DNA adducts. On-line high performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) has been used to further study the metabolites of tamoxifen formed by rat liver microsomes in the presence of NADPH with a view to identifying potential reactive metabolites which may be responsible for the formation of DNA adducts, and liver carcinogenesis. A metabolite has been detected with a protonated molecule at m/z 773. The mass of this compound is consistent with a dimer of hydroxylated tamoxifen (m/z 388). Analysis of 4-hydroxytamoxifen incubated with a rat liver microsomal preparation showed the formation of a similar metabolite with an apparent MH+ ion at m/z 773, believed to be a dimer of 4-hydroxytamoxifen formed by a free radical reaction. The retention time for this metabolite from 4-hydroxytamoxifen is identical to that of the tamoxifen metabolite, suggesting that these two compounds are the same. The levels of the dimer were higher when 4-hydroxytamoxifen was used as substrate and, in addition, two isomers were detected. It is proposed that tamoxifen was first converted to arene oxides which react with DNA or to 4-hydroxytamoxifen, either directly or via 3,4-epoxytamoxifen, which then undergoes activation via a free radical reaction to give reactive intermediates which can then react with DNA and protein, or with themselves, to give the dimers (m/z 773).

On-line high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI MS) and tandem mass spectrometry (MS/MS) have been applied to the study of tamoxifen metabolism in liver microsomes and to the identification of potentially genotoxic metabolites. The results showed that the hydroxylated derivatives, including 4-hydroxytamoxifen and alpha-hydroxytamoxifen are detoxication metabolites, while arene oxides, their free radical precursors or metabolic intermediates, are the most probable species involved in DNA-adduct formation.

An on-line high-performance liquid chromatographic (HPLC)-electrospray ionization mass spectrometric (ESI-MS) method has been developed and optimized for the study of tamoxifen metabolism. Metabolism in mouse liver microsomes was chosen to demonstrate the applicability and superiority of the method, since mice metabolize tamoxifen faster and produce more metabolites than rats or humans. Mouse liver microsomal preparations were incubated with tamoxifen in the presence of NADPH and MgCl2. The metabolites formed were separated and analyzed by the optimized HPLC-ESI-MS system. The separation was performed on a Res Elute-BD column (5 μm particle size, 250 x 4.6 mm I.D.) with 70% (v/v) methanol in 0.5 M ammonium acetate as the mobile phase. A total of eleven metabolites have been detected, some of which have not been previously reported. The metabolites identified are: tamoxifen N-oxide, N-desmethyltamoxifen, 4-hydroxytamoxifen, 4′-hydroxytamoxifen, 4-hydroxytamoxifen N-oxide, 4′-hydroxytamoxifen N-oxide, 4-hydroxy-N-desmethyltamoxifen, 4′-hydroxy-N-desmethyltamoxifen, 3,4-dihydroxytamoxifen, 3,4-epoxytamoxifen and 3,4-epoxytamoxifen N-oxide.

Seventy-nine species and three hybrids of Labiatae have been examined for the presence of betaines, which were isolated from and characterised for all the plants tested in six of the eight subfamilies (Ajugoideae, Teucrioideae, Viticoideae, Lamioideae, Pogostemonoideae and Scutellarioideae). However, unlike the Lamioideae, all species of which had relatively high betaine levels, those members tested of the other major subfamily, the Nepetoideae, either gave low betaine yields or these compounds were not detected. The three species examined representing the Chloanthoideae contained compounds which reacted with Dragendorff's reagent, but their structures could not be determined because of the small quantities isolated. The betaines found in different species of the same genus were very similar, supporting the view that these compounds have taxonomic significance at the generic level.

The separation of toremifene and its metabolites 4-hydroxytoremifene, N-desmethyltoremifene, N-desdimethyltoremifene and deaminohydroxytoremifene by reversed-phase high performance liquid chromatography is described. The effects of pH, buffer concentration and type and proportion of organic modifier on the retention and resolution of the compounds have been studied. This allows optimum conditions for a particular biological application to be developed by simple modification of these paramaters. For the separation of toremifene and metabolites in microsomal metabolism and in plasma, the optimum conditions were 65% (v/v) acetonitrile in 0.25M ammonium acetate-acetic acid buffer, pH 5.0–5.2.

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.

The metabolisms of tamoxifen in female rat, mouse and human liver microsomal preparations were compared. Rat, mouse and human liver microsomes were incubated with tamoxifen in the presence of NADPH and MgCl2 and the metabolites formed were analysed by on-line HPLC-electrospray ionization MS. The major metabolites formed by rat liver microsomes were 4-hydroxytamoxifen, 4'-hydroxytamoxifen, N-desmethyltamoxifen and tamoxifen N-oxide. In addition, two epoxide metabolites, 3,4-epoxytamoxifen and 3',4'-epoxytamoxifen, and their hydrolysed derivatives, 3,4-dihydrodihydroxytamoxifen and 3',4'-dihydrodihydroxytamoxifen, have been identified. The pattern of the main metabolites obtained with human liver microsomes resembles qualitatively that of rat liver microsomes. The major differences between rat and human liver microsomes were that the amount of hydroxylated metabolites were much lower in human and only traces of 3,4-epoxytamoxifen and the corresponding dihydrodihydroxy derivative were detected. No 3',4'-epoxytamoxifen was detected in human liver microsomes. The four major metabolites were also formed in much larger amounts and with faster rates of formation by mouse liver microsomes, though tamoxifen N-oxide clearly predominated in this species. Polar metabolites, 3,4-dihydroxytamoxifen and 4-hydroxytamoxifen N-oxide, which were undetectable in rat and human, were formed in significant amounts in mouse microsomes. As in human microsomes, there was only one epoxide metabolite, 3,4-epoxytamoxifen, produced by mouse liver microsomes at levels lower than that found in rat. The faster rate of metabolism and the production of polar metabolites may indicate the ability of mouse to detoxify tamoxifen by rapid elimination compared with rat and human. The production of a larger amount of potentially reactive epoxide metabolites in rat may be responsible for the liver carcinogenesis in this species.

The clastogenicity of tamoxifen and toremifene was tested in six human lymphoblastoid cell lines each expressing increased monooxygenase activity associated with a specific transfected human cytochrome P450 cDNA (CYP1A1, CYP1A2, CYP2D6, CYP2E1 or CYP3A4). The chemicals were also tested in a cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase, and in a cell line containing only the viral vector (Ho1). Dose-related increases in micronuclei were observed when cells expressing 2E1, 3A4, 2D6 or MCL-5 cells were exposed to tamoxifen. The positive responses in the cell lines were in the order MCL-5 > 2E1 > 3A4 > 2D6. Toremifene also gave positive results with 2E1, 3A4 and MCL-5 cells, although the responses were less marked and the positive effects required higher doses than with tamoxifen. A synthesized epoxide of tamoxifen was also tested in these cell lines and produced similar increases in the incidences of micronucleated cells. The increases in the responses observed with the epoxide were greater than with tamoxifen or toremifene. The P450 isoenzyme activities in these cells were in a range similar to those of human tumour-derived cell lines. Microsomes (1A1, 2A2, 2A6, 2B6, 2E1, 3A4 and 2D6) from these cells all metabolized tamoxifen. The major metabolite detected by HPLC was N-desmethyltamoxifen, and 4-hydroxytamoxifen was also detected in cells with cytochrome P450 2E1 and 2D6. These results are consistent with the following conclusions. (1) Tamoxifen requires metabolic activation to DNA-reactive species by specific CYP monooxygenases in order to exert its genotoxic effects. (2) The positive clastogenic effects elicited in lymphoblastoid cells by tamoxifen epoxide suggest that the genotoxic (and possibly the carcinogenic) effects of tamoxifen may be due to one or more epoxide metabolites that are generated intracellularly, probably in close proximity to the nucleus. (3) Tamoxifen is more genotoxic than toremifene.

An isocratic reversed-phase high performance liquid chromatographic method for the determination of tamoxifen and its metabolites in plasma and tissues is described. Plasma or tissue homogenate was extracted with methanol/dimethyl sulphoxide (4:1 v/v). The supernatant after centrifugation was separated on a BDS-Hypersil column with methanol/0.5 M ammonium acetate (75:25 v/v) as the mobile phase. The recoveries of tamoxifen added to plasma and liver tissue homogenate by the extraction procedure were 102 +/- 1.6 and 98 +/- 2.4% (mean +/- SD, n = 6), respectively. The solutes were detected at 280 nm with a detection limit of 0.25 micrograms/mL for tamoxifen.

From the aerial parts of Lamium maculatum L., prolinebetaine, trans -4-hydroxyprolinebetaine, pipecolic acid betaine and trans -4-hydroxypipecolic acid betaine have been isolated. The last of these compounds is a new natural product.

We investigated the metabolism of the carcinogen benzo[a]pyrene (BaP) by liver microsomes of channel catfish (Ictalurus punctatus) and brown bullhead (Ameiurus nebulosus) pretreated with 3-methylcholanthrene. The catfish liver microsomes metabolized BaP at a considerably lower rate than did the brown bullhead liver microsomes. Although the BaP metabolites produced by the liver microsomes of the two species were qualitatively similiar, there were considerable differences in the relative proportions of individual metabolites formed. The catfish liver microsomes produced a considerably lower proportion of benzo-ring diols of BaP including BaP-7,8-diol (the proximate carcinogenic metabolite of BaP) than did the bullhead liver microsomes. Compared to the bullhead liver microsomes, the catfish microsomes converted a higher proportion of BaP to phenolic metabolites.