Biomedical subjects

D R Germolec

Publications and source records attributed to D R Germolec.

At least 55 records · Page 3Linked to original sources

PubMed1995

Comparative assessment of metabolic enzyme levels in macrophage populations of the F344 rat.

The immune system is a direct target for toxic insult by a number of drugs and other chemicals, many of which require activation to toxic metabolites by drug-metabolizing enzymes. We compared the induction of drug-metabolizing enzymes, including cytochrome P450 1A1 (CYP1A1) and aldehyde dehydrogenase (ALDH), which are differentially expressed in various macrophage populations following treatment of F344 rats with the inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Kupffer cells, alveolar macrophages and splenic macrophages from TCDD-treated animals expressed elevated levels of inducible CYP1A1 as compared to other macrophage subpopulations or cells from control rats. TCDD treatment also resulted in increased ethoxyresorufin-O-deethylase (EROD) activity and total cytochrome P450 content in tissue-derived macrophages. Immunoreactive protein and mRNA transcripts for CYP1A1 were not detectable in resident peritoneal macrophages or peripheral blood monocytes. Examination of aromatic hydrocarbon receptor (AhR) levels in macrophage populations suggests that the ability of TCDD to induce metabolic enzymes in specific cell types correlates well with AhR expression. In vivo activation of macrophages, using either Bacillus of Calmette and Guérin, Mycobacterium tuberculosis (BCG) or polyinosinic:polycytidylic acid (Poly I:C), caused no significant alteration in the levels of induction of CYP1A1. ALDH-3 induction was similar in all macrophage populations examined. These studies indicate that macrophages, particularly those from portals of entry, may be induced to produce increased levels of specific enzymes, and the induction is dependent upon their maturational stage rather than their activation state. The metabolism of xenobiotics to toxic intermediates by immune cells and its role in immunosuppression are discussed.

Aldehyde Dehydrogenase↗

PubMed1995

Role of keratinocyte-derived cytokines in chemical toxicity.

Following appropriate stimulation, such as with tumor promoters, ultraviolet light or various chemical agents, keratinocytes synthesize and secrete cytokines which can mediate or participate in dermatotoxic responses such as inflammation, hyperkeratosis, hypersensitivity and skin cancer. We have determined the qualitative and quantitative cytokine response in primary human keratinocyte cultures following exposure to several non-sensitizing contact irritants, sensitizers and ulcerative agents as well as a skin carcinogen. The chemicals were also administered to mice to assess whether the dermatotoxic response correlated with the in vitro production of keratinocyte-derived cytokines. Due to the complex cellular interactions that occur in the skin, it was not possible to identify specific cytokine profiles for most of the classes of dermatotoxic agents studied. However, the non-sensitizing contact irritants produced relative increases in the synthesis and secretion of the proinflammatory cytokines, interleukin-1 and tumor necrosis factor-alpha, as well as the neutrophil chemotactic cytokine, interleukin-8 compared to the other chemical agents. While ulcerative compounds as well as irritants elicited neutrophils to the site of chemical application when applied to the mouse skin, time-dependent and chemical-specific patterns of inflammation were detected. Treatment of human keratinocyte cultures with arsenic, a human skin carcinogen, resulted in a unique cytokine profile characterized by induction of growth factors, including transforming growth factor-alpha and granulocyte-macrophage colony stimulating factor. Treatment of v-Ha-ras transgenic mice, an animal model for skin cancer, with arsenic caused an increase in the number of papillomas as well as overexpression of these growth factors suggesting that they participate in arsenic-induced skin papilloma development. These studies indicate a diverse role exists for keratinocyte-derived cytokines in dermatotoxic actions.

Animals↗

PubMed1994

Asbestos stimulates IL-8 production from human lung epithelial cells.

Studies have indicated that soluble products, including chemotactic factors, released by activated lung macrophages and fibroblasts are critical mediators in the pathogenesis of asbestos-induced pulmonary fibrosis. We provide evidence that mediators produced by lung epithelial cells in response to asbestos may also contribute to lung disease. In the present study, the carcinogenic and fibrogenic fibers, chrysotile and crocidolite asbestos, were shown to directly stimulate the human pulmonary type-II epithelial cell line, A549, and to a lesser degree primary human bronchial epithelial cells, to elicit the chemotactic cytokine IL-8 in the absence of endogenous stimuli such as IL-1 and TNF. That the membrane signaling events responsible for asbestos-induced IL-8 production are distinct from those responsible for IL-8 induction by cytokines was confirmed by using membrane-stabilizing agents and protein synthesis inhibitors. Stimulation was not observed with nonfibrogenic fibers, wollastonite and titanium dioxide, and was the direct result of asbestos-induced initiation of transcription. Asbestos failed to stimulate the release of TNF, IL-1 beta, or monocyte chemoattractant protein-1 in A549 or primary bronchial epithelial cells, indicating that cytokine secretion by asbestos is highly selective. However, a slight release of IL-1 alpha, probably preformed, was released in human bronchial epithelial cells. These data suggest that epithelial cells may, in addition to macrophages and fibroblasts, be an important effector cell in the immunopathogenesis of asbestos-associated diseases and in particular, in the neutrophilic infiltration that is commonly observed after asbestos exposure.

Asbestos, Serpentine↗

PubMed1994

Use of animal studies in risk assessment for immunotoxicology.

We have previously reported on the design and content of a screening battery involving a 'tier' approach for detecting potential immunosuppressive compounds in mice. This battery has been used to examine a variety of compounds, and the database generated from these studies, which consists of over 50 compounds, has been collected and analyzed in an attempt to improve the accuracy and efficiency of screening chemicals for immunosuppression and to identify better those tests that predict experimentally-induced, immune-mediated diseases. Specifically, these analyses attempted to develop an improved testing configuration for the accurate prediction of immunotoxic agents and to provide insight into the qualitative and quantitative relationships between a number of immune and host resistance assays commonly employed to examine potential immunotoxic chemicals in experimental animals. While a number of limitations existed in the analyses, several conclusions were drawn from the results which will be discussed.

Animals↗

PubMed1994

Development of human lymphocyte-engrafted SCID mice as a model for immunotoxicity assessment.

Mice with the severe combined immunodeficient (SCID) and triple-deficient (bg/nu/xid) mutations lack select populations of functional immune cells. Studies by several laboratories have demonstrated the ability to restore certain missing immune components in these mice by reconstituting with various lymphoid tissues including peripheral blood lymphocytes (PBL) from mice and humans. Such a model could provide an opportunity to examine human lymphoid cells in an in vivo environment for immunotoxicity assessment. In the present studies, bg/nu/xid and SCID mice were reconstituted by intraperitoneal or intravenous injection with either tetanus-immunized syngeneic mouse splenocytes (mo-SPL) or tetanus-immunized human PBLs (hu-PBL) under various test conditions. Hu-PBL-SCID mice from the C.B-17 strain produced more successful human engraftments than mice from the bg/nu/xid or C3H-SCID strains. Using optimal conditions, mo-SPL-SCID and hu-PBL-SCID mice were engrafted and administered either 2,3,7,8-tetrachlorodibenzo-p-dioxin or cyclosporin A (Cys A) and periodically bled to measure tetanus-specific antibody and class-specific immunoglobulin concentrations. Comparison of the chemical-related changes in immunoglobulin and tetanus antibody concentrations revealed some similarities between control mice and mo-SPL-SCID or hu-PBL-SCID mice, particularly with Cys A groups. However, under the various conditions examined, hu-PBL-SCID mice demonstrated considerable variability in their ability to provide consistent reconstitution, thus, limiting the ability to determine whether human cells are more or less susceptible than mouse cells to the test agents. Provided that this system can be refined to provide consistent reconstitution, hu-PBL-SCID mice may be a promising in vivo model for assessment of potential immunotoxic agents.

Animals↗

PubMed1994

Endotoxin-induced cytokine gene expression and excretion in the liver.

Peptide mediators, including tumor necrosis factor-alpha, interleukin 1 and interleukin-6, are associated with many chronic inflammatory diseases and septic shock. As such, considerable information has been collected by means of study of cytokine secretion from isolated cells or plasma cytokines during septic shock or inflammatory disorders. In this investigation, we used semiquantitative polymerase chain reaction analysis and a recently developed liver slice model to examine the characteristics of cytokine profiles that occur in the liver, the main organ involved in endotoxemia, after lipopolysaccharide challenge. Tumor necrosis factor-alpha, interleukin-1 alpha and interleukin-6 were rapidly secreted after in vivo LPS exposure or when added in vitro to rodent or human liver slice samples. This increase was associated with increased cytokine-specific mRNA transcripts. Kinetic analysis revealed that most tumor necrosis factor-alpha is released from the liver within 1 hr of lipopolysaccharide challenge, whereas interleukin-1 alpha and interleukin-6 continued to be produced for the entire culture period. Addition of monoclonal antibodies against tumor necrosis factor-alpha or interleukin-1 alpha to the culture partly inhibited interleukin-6 secretion, indicating that interleukin-1 alpha and tumor necrosis factor-alpha help mediate and sustain interleukin-6 synthesis. Depletion of hepatic sinusoidal macrophages (Kupffer cells) by a liposome-mediated macrophage "suicide" technique indicated that almost all of the secreted interleukin-1 alpha and tumor necrosis factor-alpha originate from these cells, whereas interleukin-6 secretion might also include other cell types. This study supports and extends previous findings and allows for a more rational approach to developing effective therapies against chronic inflammatory diseases and septic shock.

Alanine Transaminase↗

PubMed1993

Risk assessment in immunotoxicology. II. Relationships between immune and host resistance tests.

We have reported on the design and content of a screening battery using a "tier" approach for detecting potential immunotoxic compounds in mice (Luster et al., Fundam. Appl. Toxicol., 10, 2-19, 1988). The data base generated from these studies, which consists of over 50 selected compounds, has been collected and analyzed in an attempt to improve future testing strategies and provide information to aid in developing future quantitative risk assessment for immunotoxicity. In a recent study it was shown that as few as two or three immune parameters were needed to predict immunotoxicants in mice (Luster et al., Fundam. Appl. Toxicol., 18, 200-210, 1992). In particular, enumeration of lymphocyte populations and quantitation of the T-dependent antibody response were particularly beneficial. Furthermore, commonly employed apical measures (e.g., leukocyte counts, lymphoid organ weights) were fairly insensitive. The present analyses focus on the use of this data base to develop statistical models that examine the qualitative and quantitative relationship(s) between the immune function and host resistance tests. The conclusion derived from these analyses are: (1) A good correlation exists between changes in the immune tests and altered host resistance in that there were no instances where host resistance was altered without affecting an immune test(s). However, in some instances immune changes occurred without corresponding changes in host resistance. (2) No single immune test could be identified which was fully predictive for altered host resistance, although most assays were relatively good indicators (i.e., > 70%). Several others, such as proliferative response to lipopolysaccharide and leukocyte counts, were found to be relatively poor indicators for host resistance changes. (3) The ability to resist infectious agent challenge is dependent upon the degrees of immunosuppression and the quantity of infectious agent administered. (4) Logistic and standard regression modeling using one extensive chemical data set from the immunosuppressive agent, cyclophosphamide, indicated that most immune function-host resistance relationships followed linear rather than linear-quadratic (threshold-like) models. For most of the relationships this could not be confirmed using a large chemical data set and, thus, a more mechanistically based approach for modeling will need to be developed. (5) Using this limited data set, methods were developed for modeling the precise quantitative relationships between changes in selected immune tests and host resistance tests.

Animals↗

PubMed1993

Analyses of cytogenetic damage in rodents following exposure to simulated groundwater contaminated with pesticides and a fertilizer.

Male Fischer 344 rats and female B6C3F1 mice were each exposed through their drinking water to a mixture of pesticides and ammonium nitrate that simulated contaminated groundwater in California (California Chemical Mixture [CCM]). Exposures were for 71 or 91 days, respectively. In addition, B6C3F1 female mice were exposed for 91 days to another pesticide and ammonium nitrate mixture (Iowa Chemical Mixture [ICM]) through their drinking water. The spleens were removed from the animals, and the splenocytes were cultured for analyses of sister-chromatid exchange (SCE), chromosome aberrations (CA), and micronuclei (MN) in cytochalasin B-induced binucleate cells. A concentration-related increase in SCEs was found in the splenocytes of the rat at the 1x, 10x and 100x levels of the CCM and at the 100x concentration of the CCM in the mouse. There were no other consistent cytogenetic effects observed with the CCM, and no statistically significant cytogenetic damage was observed in mice exposed to the ICM. Evidence from the literature is discussed in order to infer which chemical or chemicals in the CCM might be responsible for the observed SCE response.

Animals↗

PubMed1992

Qualitative and quantitative experimental models to aid in risk assessment for immunotoxicology.

We have previously reported on the design and content of a screening battery using a "tier" approach for detecting potential immunosuppressive compounds in mice [1]. This battery was composed of various immune function, immunopathology and host resistance tests, the results of which could help establish the potential of chemical and biological agents to cause immunosuppression. The data from these studies, which now encompass over 50 compounds, have been analyzed in an attempt to improve future testing strategies and provide information to aid in the risk assessment process. Specifically, the following two issues will be addressed; what are the likelihood(s) for each of the individual tests and testing configurations to accurately identify immunotoxic compounds? and what are the quantitative and qualitative relationships between the immune tests and host resistance assays?

Animals↗

PubMed1992

Thymocyte injury after in vitro chemical exposure: potential mechanisms for thymic atrophy.

In addition to hepatic injury, thymic atrophy is a common observation in rodent subchronic toxicity studies. We have examined representative chemicals which produce thymic atrophy in rodents for their ability to cause direct thymocyte injury because the mechanism(s) responsible for these effects have not been determined. Although a number of the compounds examined failed to have any observable direct effect on thymocytes, others either inhibited lymphocyte proliferation or initiated cell death. In the latter group, thymocyte death was always preceded by increases in intracellular Ca++ and involved, to varying degrees, necrotic and apoptotic events. Apoptosis, as evidenced by cellular DNA cleavage into multiples of 180-200-base pair oligonucleotides and partial cell protection by cycloheximide treatment, was most evident after treatment with acetaldehyde or dibutyltin dichloride. A number of compounds that produce thymic atrophy also inhibited T lymphocyte proliferation without evidence of cell death. Considering that many of the compounds tested failed to produce any evidence of direct thymocyte injury (i.e., necrosis, apoptosis or inhibition of cell proliferation), indirect mechanisms may also be involved in thymic atrophy and may target prothymocytes in the bone marrow, after normal homing patterns or injure the thymic epithelium. Thus, it appears that a variety of mechanisms may be responsible for chemical-induced thymic atrophy and/or injury.

Animals↗

PubMed1991

Perinatal thymocyte antigen expression and postnatal immune development altered by gestational exposure to tetrachlorodibenzo-p-dioxin (TCDD).

In utero exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was found to alter expression of murine thymocyte fetal cell-surface markers. Pregnant mice were treated (via gavage) with 0, 1.5, or 3.0 micrograms TCDD/kg/day in corn oil on gestational days (gd) 6-14. Offspring were examined on gd 18 and postnatally on d6, d14, and d21, and at 7, 8, and 10 weeks of age. Severe thymic atrophy and cellular depletion were found both pre- and postnatally in TCDD-exposed mice. Immunocytochemical localization of the Thy 1.2 antigen on gd 18 thymocytes revealed no TCDD-related changes in cellular distribution. Flow cytometric analysis, however, indicated that the TCDD treatment resulted in a significant decrease in the percentage of CD4+8+ fetal thymocytes, as well as significantly increased CD4-8- and CD4-8+ thymocytes. The increased CD4-8+ population after TCDD was not from induction of Ts cells. At 7-8 weeks postnatally, no differences existed between control and treatment groups in mitogen responses and antibody plaque response. However, altered thymocyte antigen expression was found to correlate with altered postnatal immune function, as evidenced by decreased cytotoxic T lymphocyte response at 8 weeks of age. Taken together, these results indicate that immunosuppression following prenatal exposure to TCDD can be readily detected by qualitative and quantitative changes in the cell surface phenotype of fetal thymocytes. Furthermore, the observed altered distribution suggests that TCDD inhibits normal thymocyte maturational processes.

Animals↗

PubMed1991

Experimental studies of the hematologic and immune system toxicity of nucleoside derivatives used against HIV infection.

Adverse effects stemming from the therapeutic use of dideoxynucleoside derivatives continue to occur in patients with AIDS or AIDS-related complex. For example, the continued use of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-didoxycytidine (ddC), both of which confer clinical benefits in AIDS patients, may be complicated by anemia, neutropenia and thrombocytopenia and ddC also causes peripheral neuropathy. Subsequently, the National Toxicology Program (NTP) has undertaken efforts to define and characterize the toxicities associated with currently employed and potential AIDS therapeutics in experimental animals, with particular emphasis on the hematopoietic and immune systems. In addition to AZT and ddC, 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyinosine (ddI) and 2',3'-didehydrodideoxythymidine (d4T) have been examined. The present studies describe: (1) the development of a poorly regenerative macrocytic anemia in mice exposed to AZT or ddC. This anemia demonstrates a rapid and progressive time course of toxicity and reversibility after cessation of treatment; (2) the selective suppression of erythroid progenitor cells in mice exposed to d4T without concomitant effects on myeloid stem cells. Myelotoxicity appears to show metabolism-dependent strain-specificity and is more evident following in vitro exposure than in vivo exposure; and (3) the immunosuppressive effects following subchronic (30-day) exposure. Of the nucleoside derivatives studied, only ddA and ddI altered immune function and these changes were confined to suppression of antibody responses. It can be concluded that the overall similarities in the hematopoietic and immune system effects between rodents and humans indicate that such animal toxicology studies provide important information relevant to the toxicity of these drugs.

Animals↗

PubMed1991

Immune-associated toxicities induced by in vivo and in vitro exposure to interferon-alpha alone or in combination with nucleoside analogs.

The present studies examine whether immune-associated toxicities develop in rodents exposed to recombinant human interferon-alpha A/D (rHuIFN-alpha) alone or in combination with anti-retroviral nucleoside analogs. Four findings have emerged from these studies: (1) lymphocyte cell number and functional activity are suppressed after subchronic (1, 8 or 10 day) in vivo exposure to therapeutic doses of rHuIFN-alpha; (2) lymphocyte-associated toxicities lessen as in vivo exposure time to rHuIFN-alpha is extended; (3) T-cell-dependent antibody production is decreased after in vitro exposure of both antigen-specific T- and B-cells to rHuIFN-alpha; and (4) in vivo-induced leukocyte toxicities associated with either rHuIFN-alpha or the nucleoside analogs alone do not synergize when the therapies are combined. These data suggest that certain key immune parameters should be monitored carefully in the early stages of IFN-alpha therapy.

Animals↗

PubMed1991

2,3,7,8-Tetrachlorodibenzo-p-dioxin stimulation of tyrosine phosphorylation in B lymphocytes: potential role in immunosuppression.

The prototype halogenated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is carcinogenic and toxic in experimental animals. At the cellular level, TCDD toxicity is often expressed as an inhibition or alteration in normal cell maturation. In this respect, we and others have demonstrated that exposure of experimental animals to TCDD causes immunosuppression, including inhibition of B lymphocyte maturation and antibody synthesis. Although the immunological effects of TCDD are well described, little is known about its mechanism of action. In the present studies, it was found that TCDD increases membrane protein phosphorylation, which is, in part, associated with tyrosine-specific phosphorylation in B lymphocytes. This increase in phosphorylation occurred within minutes following TCDD treatment and was not associated with protein kinase C. The increase in tyrosine kinase by TCDD appears to be primarily due to de novo synthesis of new protein, because the protein synthesis inhibitors puromycin and cycloheximide, as well as the transcriptional inhibitor actinomycin D, partially inhibited the effect, although increased activity of preexisting protein cannot be fully dismissed. The dose response for increased phosphorylation by TCDD was identical to that we previously reported for inhibition of antibody synthesis, suggesting that immunosuppression by TCDD may be expressed through alterations in regulatory processes controlled by tyrosine kinases. These studies are discussed in terms of the potential role of TCDD-induced tyrosine phosphorylation in immunosuppression.

Animals↗

PubMed1990

Organ-specific hematopoietic changes induced by a recombinant human interferon-alpha in mice.

Interferon-alpha (IFN-alpha) is a naturally occurring cytokine that mediates numerous biological activities and has demonstrated therapeutic potential in a variety of malignancies. Encouraging activity against HIV-1 replication has also been observed with IFN-alpha in the treatment of AIDS, although hematotoxicity has been a frequently observed side effect. In addition, in vitro studies have suggested that IFN-alpha may function as a down-regulator of myelopoiesis. A recombinant hybrid of subtypes of human IFN-alpha, rHuIFN-alpha A/D, has antiviral activity in murine cells in vitro and in vivo. This study examines the effect of acute and subchronic exposure to rHuIFN-alpha A/D on hemopoietic and immune parameters in C57Bl/6 mice. IFN-alpha was administered ip at 0, 1000, 10,000, and 100,000 units/day for either 1 or 10 consecutive days. Many of the known effects of IFN-alpha in humans such as anemia, leukopenia, and thrombocytopenia were observed in mice following subchronic exposure, with the latter two effects also manifested following acute exposure. Further analysis showed that this leukopenia was not selective. Both splenic and bone marrow cells were examined following 10 days of dosing with the high dose of IFN-alpha. Lymphocytes were reduced in both compartments, while granulocytes were increased in both compartments. Bone marrow cells programmed to differentiate into granulocytes (CFU-G) were suppressed, while macrophage progenitors (CFU-M) were stimulated. Erythroid cells decreased in the marrow but increased in the spleen, suggesting that the microenvironment may play a significant role in the effect of IFN-alpha. The proliferative capacity of both B and T splenic lymphocytes was significantly suppressed in a dose-related fashion following multiple exposure to IFN-alpha. Clinically, IFN-alpha is most often given in multiple doses and the present data suggest that such a regimen is toxic to both erythroid and myeloid cells, as well as being immunotoxic to splenic B and T lymphocytes.

Alanine Transaminase↗

PubMed1990

Panax ginseng as a potential immunomodulator: studies in mice.

There has been continuing interest in the development of synthetic and natural compounds which modify the immune response, particularly for the treatment of AIDS and cancer. Panax ginseng, employed for its putative medicinal properties in South Asia, was examined for its immunomodulatory properties in mice. A systematic evaluation of multiple immune system components revealed that Panax ginseng stimulated basal natural killer (NK) cell activity following subchronic exposure and helped stimulate recovery of NK function in cyclophosphamide-immunosuppressed mice but did not further stimulate NK activity in poly I:C treated mice. Other immunological parameters examined, including T and B cell responses were not affected. Panax ginseng provided a degree of protection against infection with L. monocytes but did not inhibit the growth of transplanted syngeneic tumor cells. Increased resistance to L. monocytogenes was not detected in challenged mice previously given immunosuppressive doses of cyclophosphamide. Taken together, these data suggest that Panax ginseng has some immunomodulatory properties, primarily associated with NK cell activity.

Adjuvants, Immunologic↗

PubMed1990

Immunotoxicology: review of current status.

Our need to understand the potential adverse human health effects of environmental chemical exposure has coincided with an increased understanding of the immune system and an appreciation of its complex regulatory network. This has spawned a broad interest in the area of immunotoxicology within the scientific community as well as certain concerns in the public sector regarding chemical-induced hypersensitivity and immunosuppression. The incidence of alleged human sensitization to chemicals has increased, in part, due to the fact that chemical companies are moving to larger and/or different markets. It has been estimated that 35 million Americans suffer from allergic disease of which 2% to 5% are from occupational exposure. Although there is not yet a clear understanding of dose-response relationships or disease predisposition, there are many well-defined examples (isocyanates, anhydrides) of chemical sensitizers in humans and experimental animals. Evidence that chemicals suppress immune responses in humans is considerably less well established, although there is a public perception that chemicals generally cause immunosuppression. This perception has been fueled by highly publicized legal cases (ie, W.R. Grace and Agent Orange) and scientific controversies within the academic and industrial communities (Aldicarb). As a consequence of public pressure, the regulatory agencies are considering immunotoxicity testing guidelines. At the present, however, there are limitations on adequate human methodology and data that allow the extrapolation of animal data to assess accurately human risk.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

12S,19- and 12S,20-dihydroxyeicosanoids: novel 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid metabolites formed by hydroxylation and reduction in murine lymphocytes.

Murine spleen cells and purified B lymphocytes oxidized arachidonic acid via the lipoxygenase pathway. The major metabolite of both the whole spleen and enriched B lymphocytes was 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid. A novel metabolite was observed that did not have an absorbance from 210 to 400 nm, indicating the absence of a conjugated double bond system. The new metabolite was converted to the methyl ester, reduced by platinum oxide, derivatized to the trimethylsilyl ether, and analyzed by gas chromatography-mass spectrometry. A major and a minor component were observed in the analysis of the new compound. The major component had major diagnostic ions indicating the presence of hydroxyl groups at C-12 and C-19. The minor component had major diagnostic ions indicating the presence of hydroxyl groups at C-12 and C-20. The new metabolites are characterized as a mixture of 12S,19- and 12S,20-dihydroxyeicosanoids presumably formed by hydroxylation and reduction of one or more double bonds of 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid. These metabolites were formed predominantly with whole spleen lymphocytes but could be detected at longer incubation times or by using 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid as the starting substrate with highly enriched B lymphocytes.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗