Characterization of micronuclei induced in human lymphocytes by benzene metabolites

Benzene is an established human leukemogen. Workers occupationally exposed to benzene exhibit increased frequencies of both structural and numerical chromosomal aberrations in their peripheral blood lymphocytes. The metabolite(s) responsible for these chromosomal aberrations has not yet been identified. Using a modified micronucleus assay, we have examined the ability of the metabolites of benzene to induce chromosomal damage in human lymphocytes. An antikinetochore antibody was used to distinguish micronuclei that have a high probability of containing a whole chromosome (kinetochore positive) from those containing acentric fragments (kinetochore negative). In vitro treatments with the benzene metabolites hydroquinone, 1,4-benzoquinone, phenol, and catechol resulted in significant increases in micronuclei formation. Phenol, catechol, and 1,4-benzoquinone treatments resulted in moderate (2- to 5-fold) increases in micronuclei, whereas hydroquinone treatments resulted in a larger (11-fold) increase in micronuclei. Significant dose-related increases in kinetochore-positive micronucleated cells were not observed following 1,4-benzoquinone treatment but were observed following treatment with phenol, catechol, and hydroquinone. The higher efficacy of hydroquinone in inducing both total micronuclei and kinetochore-positive micronucleated cells when compared with catechol, phenol, and 1,4-benzoquinone suggests that hydroquinone is a major contributor to the clastogenicity and aneuploidy observed in the lymphocytes of benzene-exposed workers. Other metabolites may also contribute, however, to the genotoxic effects of benzene. Since consistent chromosomal aberrations are often observed in human leukemias, the ability of the phenolic metabolites of benzene to induce chromosomal damage in human cells also implicates them in benzene-induced leukemia.     

In vitro and in vivo MRS study of human glioma metabolites

In this study an in vitro multinuclear magnetic resonance spectroscopy (MRS) characterization of water soluble metabolites and of the lipid fraction obtained from 19 human gliomas (12 glioblastomas, 3 anaplastic astrocytomas, 2 anaplastic oligodendrogliomas and 2 oligodendrogliomas) with a total of 27 surgical specimens, is reported. Moreover, some in vivo H-1 MRS results are shown. The regional metabolic heterogeneity of glioblastomas, according to their morphological heterogeneity, is documented. For glioblastomas a specific in vitro H-1 MRS metabolite profile cannot be defined. Low and high grade oligodendrogliomas showed characteristic choline/creatine and alanine/creatine metabolite ratios. The spectroscopical characterization of histopathological factors concerning malignant gliomas is shown.     

Inhibition of interferon gamma production by benzene and benzene metabolites

We exposed spleen cells from female Swiss/Webster mice to benzene and benzene metabolites to determine the effects of such exposure on interferon gamma induction by concanavalin A. Exposing the cells to benzene or phenol did not affect interferon gamma production, but exposing them to p-benzoquinone, catechol, or hydroquinone significantly inhibited interferon gamma production. Cell viability, as determined by trypan blue viability staining, was not influenced by the chemical treatment. When interferon gamma production was inhibited, the inhibition was dose dependent. The time of optimum production of interferon gamma after exposure to concanavalin A was not affected by treatment of the cells with p-benzoquinone. These data indicate the importance of dihydroxy and diketo metabolites as immunotoxic derivatives of benzene.     

Sister chromatid exchange induction in human lymphocytes exposed to benzene and its metabolites in vitro

Previous in vivo studies have shown that low-dose benzene exposure (10 to 28 ppm for 4 to 6 h) in mice can induce sister chromatid exchange (SCE) in peripheral blood B-lymphocytes and bone marrow as well as micronuclei in bone marrow polychromatic erythrocytes. Because benzene is metabolized to a variety of intermediate compounds and two of these, catechol and hydroquinone, have been reported to be potent SCE-inducers, it is possible that other known and proposed metabolites could have chromosome-damaging effects in lymphocytes. Induced SCE frequencies, mitotic indices, and cell cycle kinetics were quantitated in human peripheral blood T-lymphocytes exposed to benzene, phenol, catechol, 1,2,4-benzenetriol, hydroquinone, 1,4-benzoquinone, or trans,trans-muconic acid. Three proposed metabolites of phenol, 4,4'-biphenol, 4,4'-diphenoquinone, and 2,2'-biphenol, which can be generated by a phenol-horseradish peroxidase-hydrogen peroxide system were also examined. Benzene, phenol, catechol, 1,2,4-benzenetriol, hydroquinone, and 1,4-benzoquinone induced significant concentration-related increases in the SCE frequency, decreases in mitotic indices, and inhibition of cell cycle kinetics. Based on the slope of the linear regression curves for SCE induction, the relative potencies were as follows: catechol greater than 1,4-benzoquinone greater than hydroquinone greater than 1,2,4-benzenetriol greater than phenol greater than benzene. On an induced SCE per microM basis, catechol was approximately 221 times more active than benzene at the highest concentrations studied. trans,trans-Muconic acid had no significant effect on the cytogenetic parameters analyzed. 2,2'-Biphenol induced a significant increase in SCE only at the highest concentration analyzed, and 4,4'-biphenol caused a significant increase in SCE frequency that was not clearly concentration related. However, both 2,2'- and 4,4'-biphenol caused significant cell cycle delay and mitotic inhibition. 4,4'-Diphenoquinone caused only a significant decrease in mitotic activity. These data indicate that in addition to phenol, di- and trihydroxybenzene metabolites play important roles in SCE induction. Furthermore, the results suggest either that benzene alone can induce SCE or, a more likely possibility, that mononuclear leucocytes have a limited capability to activate benzene.     

Clinical pharmacokinetics of navelbine after oral administration, in vitro metabolism and interindividual variability

Navelbine (NVB) (5'-noranhydrovinblastine) is a new semi-synthetic vincaalkaloid (VA) exhibiting a high affinity for tubulin and considerable anticancer activity in patients. A better hematologic tolerance and a weak neurotoxicity have been reported for this drug as compared to other VA. Moreover, NVB presents a relatively high bioavailability and a good digestive tolerance, thus offering original perspectives for the treatment of ambulatory cancer patients. A clinical pharmacokinetic study of NVB was carried out on 12 patients after oral administration of the drug. The pharmacokinetic parameters were similar to those of intravenous administration and also showed a high interindividual variability. Studies on the in vitro metabolism of NVB using hepatic microsomal fractions from 22 different donors demonstrated the formation of 3 metabolites. The biotransformation rate quantitatively varies from one human liver specimen to another, a fact which could be, in part, at the origin of the interindividual variability of the therapeutic response.     

Cytokines as potential biomarkers of liver toxicity

Several important drug classes show pre-clinical hepatotoxicity or, in some cases hepatotoxicity in man in Phase III/IV not predicted by pre-clinical studies. This hepatotoxicity is associated with death of the parenchyma by both necrosis and apoptosis. Recent data have implicated molecular mediators of the immune response such as tumor necrosis factor alpha (TNFalpha), interleukin 1beta(1L-1beta) and interleukin 6 (IL6) in acute and chronic liver damage. These cytokines networks have been implicated in mediating the hepatic response to xenobiotics as diverse as PPAR ligands, acetaminophen and phenobarbitone. Thus, pro-inflammatory cytokines such as TNFalpha, IL1 beta and IL6 are released into the bloodstream both from the liver and from distal sites during hepatic toxic injury. Probably due to differences in the responses of rodent and human hepatocytes to cytokines, some clinical hepatotoxicities are not predicted by rodent models. However, the cytokine changes implicated in this human hepatic cell death could be manifest in rodent models and thus could be detected at the molecular level. Here we review the role of cytokines in different types of drug-induced liver injury and discuss whether these cytokine fingerprints are potential biomarkers of so-called idiosyncratic human liver toxicity.     

In vitro metabolism of o-aminoazotoluene and mutagenesis of Salmonella by the metabolites

Incubation of hepatocarcinogenic aminoazo dye, o-aminoazotoluene(OAT) with rat liver microsomes together with NADPH and NADHyielded N-hydroxy-OAT (I), 4' -hydroxy-OAT (II) and a smalleramount of 2' -hydroxymethyl-3-methyl-4-aminoazobenzene (III).As an artifact 4, 4'-bis(otolylazo)-2, 2' -dimethylazoxybenzene(IV) was also detected in a small quantity. The mutagenicitiesof these metabolites were assayed by using Salmonella typhimuriumTA98 and TA100 together with S-9 prepared from the livers ofrats treated with polychlorinated biphenyl mixture (PCB). OATand III were strongly mutagenic, but only when S-9 was present.In contrast, I was a strong mutagen regardless of the presenceor absence of S-9. II and IV were non-mutagenic. The yieldsof I, II and III from OAT were pronouncedly reduced by additionof cytochrome P-450 inhibitors, especially by a cytochrome P-448inhibitor 7,8-benzoflavone. Mutagenesis by OAT was also inhibitedby addition of 7,8-benzoflavone. Activation of OAT for mutagenesiswas enhanced by pretreatment of the donor rats with PCB or 3-methyl-cholanthreneand to a much lesser extent by phenobarbital. These findingssuggest that N-hydroxylation of OAT mainly proceeds via catalysisby cytochrome P-448 and that this process is an obligatory stepfor the activation of OAT. Synthetic methods for the preparationof new azo compounds such as I, IV and 2', 3-dimethyl-4-nitrosoazobenzeneare described.     

In vitro clonogenic growth and metastatic potential of human operable breast cancer

In 254 patients with operable [International Union against Cancer (1972) Stages I, II, and III] breast cancer, the relations between in vitro clonogenic growth in soft agar of primary breast cancer tumors and their metastatic potential expressed by the relapse-free survivals (RFS) as well as overall survivals were studied. Sixty-four % (163 of 254) of cancers formed distinct colonies (30 or more colonies in a single dish, or 10 or more colonies in plural dishes). Other breast cancers (36%, 91 of 254) were designated to be negative for the clonogenicity. There was no correlation between the positive or negative clonogenicity and clinicopathological characteristics in breast cancer patients, including the age of patients, menopausal status, tumor size, T classification, International Union against Cancer stage, histological type (Japanese Breast Cancer Society), histologically proved axillary lymph node metastasis, and estrogen receptor (ER). At the time of median follow-up of 43 mo (range, 25 to 61 mo) after mastectomy, a recurrence of malignancy occurred in 19.0% (31 of 163) of the patients with positive clonogenic tumors, and in 8.8% (8 of 91) of those with negative clonogenic tumors (P = 0.03). There also was a significant difference (P less than 0.03 by log rank test, P less than 0.05 by generalized Wilcoxon test) in RFS curves between positive and negative clonogenicity groups. These differences in RFS were also noted in Stage II patients in favor of the negative colony formation group. In ER-negative cancer patients, the RFS of patients with positive clonogenic cancers was shown to be worse (P less than 0.03 by log rank test, P less than 0.05 by generalized Wilcoxon test) than patients with negative clonogenic cancers. There was no difference in RFS in ER-positive cancer patients. There was a trend (P = 0.09 by log rank test) of worse overall survival rate in patients with positive clonogenic growth. In a multivariate comparison using the principal component analysis composed of factors including positive node, T classification, histological type, age, ER, and colony formation, the clonogenicity showed a significant effect on the recurrence of malignancy and also on the survival of the patients after mastectomy. In conclusion, in vitro clonogenic growth of the primary tumor of breast cancer was shown to be one of the independent factors of metastatic potential in operable breast cancer patients after mastectomy.     

In vitro metabolism by mouse and human liver preparations of halomon, an antitumor halogenated monoterpene

Objectives: To characterize the enzymes responsible for and metabolites produced from the metabolism of halomon, a halogenated monoterpene that is isolated from the red algae Portieria hornemanii and has in vitro activity in the NCI screen against brain, renal, and colon cancer cell lines. Materials and methods: Mouse and human liver fractions, prepared by homogenization and differential centrifugation, were incubated with halomon, extracted with toluene, and analyzed by gas chromatography. Results: In the presence of NADPH, mouse-liver 9,000-g supernatant (S9) fractions metabolized halomon, but boiled S9 fractions did not. NADH could not substitute for NADPH. Further separation of murine hepatic S9 fractions produced a microsomal fraction that contained all of the halomon-metabolizing activity; cytosol had none. Carbon monoxide reduced murine hepatic microsomal metabolism of halomon, whereas an anaerobic, N₂ environment greatly accelerated the disappearance of halomon. Human hepatic microsomes metabolized halomon and required NADPH to do so. Carbon monoxide completely inhibited human hepatic microsomal metabolism of halomon. Unlike murine hepatic microsomal metabolism of halomon, anaerobic conditions did not enhance the metabolism of halomon by human hepatic microsomes. Neither 100 μM diethyldithiocarbamate, 1 μM quinidine, 100 μM ciprofloxacin, 3 μM ketoconazole, nor 100 μM sulfinpyrazone inhibited the metabolism of halomon by human hepatic microsomes. Both murine and human hepatic microsomes produced a metabolite of halomon. The mass spectrum of this metabolite indicated the loss of one chlorine atom and one bromine atom. Conclusions: Halomon is metabolized by mouse and human hepatic cytochrome P-450 enzymes, the identities of which remain unknown. Hepatic metabolism of halomon is very consistent with the concentrations of halomon measured in mouse tissues and urine after i.v. administration of the drug. Received: 23 February 1997 / Accepted: 9 June 1997     

In vitro metabolism of benzo(a)pyrene by human liver microsomes and lymphocytes.

The metabolism of benzo(a)pyrene by human liver microsomes and human lymphocytes has been analyzed by high-pressure liquid chromatography. Human liver forms seven known metabolites and at least five additional unidentified metabolites that migrate as distinct peaks. Lymphocytes incubated with benzo(a)pyrene for 30 min do not form dihydrodiols. Lymphocytes incubated for 24 hr with benzo(a)pyrene form all of the metabolites produced by liver including dihydrodiols as well as additional metabolites. The ratios of phenols formed by liver and lymphocytes are different, and preparations from humans form a different profile of metabolites than that formed by rat liver.     

The role of nitric oxide on DNA damage induced by benzene metabolites

Benzene, a tobacco constituent, is a leukemogen in humans and a carcinogen in rodents. Several benzene metabolites generate superoxide anion (O(2)(.-)) and induce nitric oxide synthase in the bone marrow of mice. We hypothesized that the reaction of nitric oxide (*NO) with O(2)(.-) leads to the formation of peroxynitrite as an intermediate during benzene metabolism. This hypothesis was supported by demonstrating that the exposure of mice to benzene produced nitrated metabolites and enhanced the levels of protein-bound 3-nitrotyrosine in the bone marrow of mice in vivo. In the current study, we investigated the influence of nitric oxide, generated from sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, on DNA strand breaks induced by each single or binary benzene metabolite at different doses and compared the levels of the DNA damage induced by each benzene metabolite in the presence of nitric oxide with the levels of DNA strand breaks induced by peroxynitrite at similar doses in vitro. We found that among benzene metabolites only 1,2,4-trihydroxybenzene (BT) can induce significant DNA damage in the absence of nitric oxide. While 1,4-dihydroxybenzene (HQ), 1,4-benzoquinone (BQ) and 1,2-dihydroxybenzene (CAT) require .NO to induce DNA strand breaks, hydroquinone was the most potent DNA-damaging benzene metabolite in the presence of *NO. The order of DNA breaks by benzene metabolites in the presence of *NO is: Peroxynitrite = HQ > BT > BQ > CAT. The *NO and O(2)(.-) scavengers inhibited DNA damage induced by [HQ+*NO]. Benzene, trans,trans-muconaldehyde, and phenol, do not induce DNA strand breaks either in the absence or presence of *NO. However, adding phenol to [HQ+*NO] leads to greater DNA damage than [HQ+*NO] alone. Collectively, these results suggest that nitric oxide is an important factor in DNA damage induced by certain benzene metabolites, probably via the formation of the peroxynitrite intermediate. Phenol, the major benzene metabolite that does not induce DNA damage alone and is inactive in vivo, synergistically enhances DNA damage induced by potent benzene metabolite in the presence of nitric oxide.     

Identification of benzene oxide as a product of benzene metabolism by mouse, rat, and human liver microsomes

Benzene is a ubiquitous environmental pollutant that is known to cause hematotoxicity and leukemia in humans. The initial oxidative metabolite of benzene has long been suspected to be benzene oxide (3,5- cyclohexadiene-1,2-oxide). During in vitro experiments designed to characterize the oxidative metabolism of [14C]benzene, a metabolite was detected by HPLC-radioactivity analysis that did not elute with other known oxidative metabolites. The purpose of our investigation was to prove the hypothesis that this metabolite was benzene oxide. Benzene (1 mM) was incubated with liver microsomes from human donors, male B6C3F1 mice, or male Fischer-344 rats, NADH (1 mM), and NADPH (1 mM) in 0.1 M sodium phosphate buffer (pH 7.4) and then extracted with methylene chloride. Gas chromatography-mass spectrometry analysis of incubation extracts for mice, rats, and humans detected a metabolite whose elution time and mass spectrum matched that of synthetic benzene oxide. The elution time of the benzene oxide peak was approximately 4.1 min, while phenol eluted at approximately 8 min. Benzene oxide also coeluted with the HPLC peak of the previously unidentified metabolite. Based on the 14C activity of this peak, the concentration of benzene oxide was determined to be approximately 18 microM, or 7% of total benzene metabolites, after 18 min of incubation of mouse microsomes with 1 mM benzene. The metabolite was not observed in incubations using heat- inactivated microsomes. This is the first demonstration that benzene oxide is a product of hepatic benzene metabolism in vitro. The level of benzene oxide detected suggests that benzene oxide is sufficiently stable to reach significant levels in the blood of mice, rats, and humans and may be translocated to the bone marrow. Therefore benzene oxide should not be excluded as a possible metabolite involved in benzene-induced leukemogenesis.      

Inhibition of interferon-alpha/beta induction in L-929 cells by benzene and benzene metabolites

Murine L-929 cells were treated with benzene or a series of benzene metabolites, washed and then interferon-alpha/beta was induced with polyriboinosinic-polyribocytidylic acid. Exposure of the cells to benzene or phenol, a monocyclic metabolite of benzene, did not affect interferon-alpha/beta induction. However, exposure of the cells to p-benzoquinone, hydroquinone or catechol, dihydroxy- and diketo-metabolites of benzene, resulted in a severe inhibition of interferon-alpha/beta production. There was no significant loss of viability of the cell cultures. Additional studies with p-benzoquinone indicated that inhibition of interferon-alpha/beta was reversible and could be abrogated by addition of reduced glutathione to the cell cultures.     

In vitro stimulation of human breast tissue by human prolactin.

Normal human breast tissue was cultured with defined media plus hormones. The epithelium survived at least 4 days in culture but did not grow in the absence of hormones. Both insulin and human prolactin stimulated growth, but ovine prolactin did not.     

In vitro cariogenic potential of Candida albicans

The adherence and dissociation of Candida albicans, C. tropicalis, Streptococcus mutans and S. sanguis to six substrates including hydroxylapatite (HAP) which exhibit various hydrophobicity, was examined by the use of a bioluminescent adenosine triphosphate (ATP) assay. Dissolution of HAP by C. albicans or S. mutans was determined spectrophotometrically by the use of o-cresolphthalein complexone. In the adherence of C. tropicalis, S. mutans and S. sanguis, the amount of adherent cells correlated with the hydrophobicity of the substrates. In contrast, the adherence of C. albicans to HAP was extraordinary high, although the adherence of the fungi also correlated with the hydrophobicity of the substrates, except for HAP. The yeasts attached to HAP was effectively removed by high concentration of either phosphate or calcium ions. The amount of calcium-release from HAP caused by C. albicans and S. mutans was 113 microg ml(-1) (final pH = 3.45), and 5.4 microg ml(-1) (final pH 4.81), respectively and the maximum growth of C. albicans and S. mutans was 10(7) cfu ml(-1) and 7.4 x 10(12) cfu ml(-1), respectively. The results, taken together, suggest that C. albicans adhere to HAP specifically through electrostatic interaction, and that, in a much smaller number (1.0/7.4 x 10(5)), C. albicans possesses the ability to dissolve HAP to a greater extent (approximately 20-fold) when compared with S. mutans.     

In vitro andin vivo modulation of growth regulation in the human breast cancer cell line MCF-7 by estradiol metabolites

Background  The natural estrogen 17β-estradiol (E₂) functions as a potent tumor promoter during tumorigenic transformation of the mammary gland. From amongst the various pathways of E₂ metabolism upregulation of C16α-hydroxylation of E₂ has been associated with carcinogenesis. In the present studyin vitro andin vivo experiments were performed on estrogen receptor positive human breast cancer MCF-7 cells to examine whether the natural estrogen E₂ and its metabolites 16α-hydroxyestrone (16α-OHEi) and 2-hydroxyestrone (2-OHE₁) function as modulators of tumor cell growth. Methods  An anchorage-independent growth assay was used forin vitro study by counting the number of tri-dimensional colonies formed by MCF-7 cells suspended in 0.33% agar.In vivo experiments examined the effect of implanting metabolite material pellets into female nude mice. Results  In the anchorage-independent growth assay (AIG), continuous 14-day exposure to E₂ and to 16α-OHE₁ at 200 ng/ml induced a 59.4% and a 105.9% increase (P=0.001) respectively in the number of colonies of MCF-7 cells. Identical treatment with 2-OHE₁, however, failed to increase AIG relative to that seen in the solvent treated control cultures. In thein vivo tumorigenicity assay, treatment of nude mice with 1.5 mg E₂ or 16α-OHE₁ resulted in a 335.4% and a 384.1% increase (P<0.0002) in tumor growth, while identical treatment with 2-OHE₁ failed to exhibit any increase relative to the control group. Conclusions  These results suggest that the 16α- and 2-hydroxylated metabolites of E₂ may directly affectin vitro growth of MCF-7 cells via an autocrine mechanism andin vivo growth via paracrine mechanisms. Thus, E2-mediated growth regulation in MCF-7 cells may in part be due to distinct effects of specific E₂ metabolites on the breast cancer cells.     

In vitro studies on the influence of doxorubicin in combination with recombinant interferon-gamma on human monocytes.

The maturation and differentiation process of human monocytes and human monocyte-derived macrophage cytotoxicity mediated by receptors for the Fc moiety of immunoglobulin G (Fc gamma Rs) were studied in vitro using the chemotherapeutic drug doxorubicin in combination with the biological response modifier (BRM) recombinant interferon-gamma (rIFN-gamma). Human monocytes were cultured for 9 days and treated with doxorubicin before, after, or on day 7 of the culture. rIFN-gamma was added continuously on day 0 or on day 7 of the culture, either alone or in combination with doxorubicin. In the presence of doxorubicin, all measured intracellular enzyme activities were at the same level as the controls and the rIFN-gamma treated cells. However, when monocytes were co-cultured for 9 days with rIFN-gamma alone or in combination with doxorubicin, enzyme levels decreased to between 45% and 61% of the controls. In control cultures ADCC activities against maximally sensitized hRBC mediated by Fc gamma RI and Fc gamma RII were 41.7 +/- 8.8% and 42.7 +/- 11.6%, respectively. When monocytes were exposed to rIFN-gamma continuously or 40 h before harvesting, Fc gamma RI ADCC activity increased to 78.9 +/- 9.8% and 68.1 +/- 8.1%, respectively, and Fc gamma RII ADCC activity to 56.3 +/- 8.4% and 55.4 +/- 7.3%, respectively. The addition of doxorubicin to monocyte cultures in the presence or absence of rIFN-gamma did not influence the lysis of the two types of sensitized hRBC. These observations indicate that doxorubicin does not negatively influence the activation state of monocytes/macrophages, induced by rIFN-gamma.