Gas chromatographic determination of catecholestrogens following isolation by solid-phase extraction.

A sensitive and specific assay for the determination of the catecholestrogens 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) using gas chromatography with electron-capture detection (GC-ECD) is described. The formation of 2- and 4-OHE2 was assessed following activation of 17beta-estradiol in the microsomal fraction of female rat livers. The analytes were isolated by solid-phase extraction, derivatized to their heptafluorobutyryl esters with heptafluorobutyric acid anhydride, and subjected to solvent exchange prior to analysis; this resulted in minimal chromatographic interference, long column life, and stable derivatized analytes. Derivatized catechols were separated and confirmed with dual column chromatography (DB-5 and DB-608) and quantitated using GC-ECD. The DB-608 column was preferred for quantitation as it provided better 4-OHE2 resolution from interference. Key validation parameters for the assay include sensitivity, intra- and inter-assay precision, and accuracy. Instrument sensitivity and limits of detection (LOD) and quantitation (LOQ) were determined statistically from fortification data approaching expected limits. For 2-OHE2 and 4-OHE2, respective values for these parameters were; instrument sensitivities of 0.4 and 0.7 pg, LODs of 0.8 and 1.3 ng/mg, and LOQs of 2.6 and 4.3 ng/mg.

Disruption of mitochondrial respiration by melatonin in MCF-7 cells.

Clinical and laboratory studies have provided evidence of oncostatic activity by the pineal neurohormone melatonin. However, these studies have not elucidated its mechanism of action. The following series of MCF-7 breast tumor cell studies conducted in the absence of exogenous steroid hormones provide evidence for a novel mechanism of oncostatic activity by this endogenous hormone. We observed a 40--60% loss of MCF-7 cells after 20-h treatment with 100 nM melatonin, which confirmed and extended previous reports of its oncostatic potency. Interestingly, there were no observed changes in tritiated thymidine uptake, suggesting a lack of effect on cell cycle/nascent DNA synthesis. Further evidence of a cytocidal effect came from morphologic observations of acute cell death and autophagocytosis accompanied by degenerative changes in mitochondria. Studies of mitochondrial function via standard polarography revealed a significant increase in oxygen consumption in melatonin-treated MCF-7 cells. Enzyme-substrate studies of electron transport chain (complex IV) activity in detergent permeabilized cells demonstrated a concomitant 53% increase (p < 0.01) in cytochrome c oxidase activity. Additional studies of succinate dehydrogenase activity (complex II) as determined by reduction of (3-4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide demonstrated a significant increase (p < 0.05) in melatonin-treated cells and further confirmed the accelerated ET activity. Finally, there was a 64% decrease (p < 0.05) in cellular ATP levels in melatonin-treated cells. The G-protein-coupled melatonin receptor antagonist luzindole abrogated the cytotoxic and mitochondrial effects. These studies suggest a receptor-modulated pathway of cytotoxicity in melatonin-treated MCF-7 tumor cells with apparent uncoupling of oxidative phosphorylation.

Kinetics of metallothionein gene induction by cadmium in human lymphocytes.

Induction of gene transcription is a complex process involving a diverse set of transcription factors and regulatory steps. We have taken a kinetic approach to analysis of metallothionein gene induction in human peripheral blood lymphocytes. By repeated measurements of MT mRNA after incubation of cells in vitro with CdCl2, we were able to determine individual-specific time related constants. The kinetics of induction for 3 individuals followed an S shaped curve and the data was fitted to a modified kinetic model of gene transcription. From this model, which assumes a cooperativity effect, transcriptional and RNA degradation rate constants could be calculated. The rate constant for transcription was doubled with the doubling of CdCl2 concentration, but the rate constant for RNA degradation was independent of Cd concentration.

Functional significance of different human CYP1A1 genotypes.

At least two different polymorphisms in the human CYP1A1 gene have been associated with an increased risk for tobacco-related lung cancer; however, the functional significance of these polymorphisms has not been determined. We measured CYP1A1 genotypes, gene expression levels and enzymatic activity levels in mitogen-stimulated lymphocytes to determine whether genetic polymorphisms in CYP1A1 alter transcriptional and/or post-transcriptional regulation of the gene. Genotypes were determined at two sites previously associated with lung cancer: a point mutation in exon 7 near the catalytic region of the enzyme and an Msp1 RFLP in the 3' non-coding region of the gene. Variant genotypes at the Msp1 site had no effect on CYP1A1 gene induction, however, variant genotypes at the exon 7 site were significantly associated with increased CYP1A1 gene inducibility. We also observed a significant interaction between the exon 7 polymorphism and smoking on mRNA levels. There was a 3-fold elevation in CYP1A1 enzymatic activity in exon 7 variant genotypes. When Msp1 and exon 7 genotypes were combined, there was an increased CYP1A1 inducibility and enzymatic activity in subjects with the exon 7 polymorphism, and in subjects with both polymorphisms.

A novel CYP1A1 gene polymorphism in African-Americans.

A new Msp1 RFLP in the CYP1A1 gene has been found in genomic DNA from African-Americans. The polymorphism results from a single A-T to G-C transition in the 3' noncoding region approximately 300 bp upstream from the polyadenylation site. This mutation leads to cleavage of the normal 2.3 kb MspI restriction fragment into 1.3 and 1.0 kb fragments. The heterozygous mutation has been seen in 8 of 47 African-Americans, but was not detected in 191 Caucasians or 30 Asians. No linkage was observed with either of the two previously described polymorphisms in this gene.

The Malmö biomarker programme.

Evaluation of pro-oxidant/antioxidant dietary factors in relation to individual susceptibility to cancer and cardiovascular disease is justified based on a review of the literature. This working hypothesis is amendable to further scientific validation from biomarkers with end-point sensitivity to oxygen radicals. So far, the biomarker programme developed around this theme may be divided into three distinct classes: (1) Markers of genotoxic exposure estimate DNA damage either directly as a biologically effective dose, or indirectly by estimating aberrant cellular functions that lead to accumulation of DNA damage. The examples included are ADP-ribosylation in mononuclear leucocytes (R. Pero, A. Olsson), oxidative DNA damage (K. Frenkel), gene expression in lymphocytes (S. Garte, G. Cosma), serum alpha macroglobulin (W. Troll) and oxidized DNA damage and repair (N. Christie). (2) Markers of genetic predisposition have been shown to have genetic inheritance patterns that relate to individual susceptibility to cancer or cardiovascular disease. The examples included are glutathione transferase mu phenotyping (R. Pero, J. Seidegård) and poly (ADP-ribose) polymerase pseudogene polymorphism (M. Smulson). (3) Markers of dietary status have been validated to estimate the amount of a particular nutrient or xenobiotic in the diet that has been taken up and metabolized or distributed to body fluids or tissues. The example included here is niacin nutriture (E. Jacobson, M. Jacobson). This biomarker is presented in Section 5 (pp. 59-62) of the Malmö Diet and Cancer Programme Minisymposium reported in this issue of the journal.

Expression of the CYP1A1 gene in peripheral lymphocytes as a marker of exposure to creosote in railroad workers.

We have conducted a pilot study to assess levels of cytochrome CYP1A1 gene expression in human peripheral lymphocytes as a molecular biomarker assay for polycyclic hydrocarbon exposure. Basal and 3-methylcholanthrene-induced levels of gene expression were measured by standard slot-blot mRNA analyses in mitogen-stimulated cultures of peripheral blood lymphocytes from creosote-exposed railroad workers and unexposed control subjects. Dermal and inhalation exposure of workers to creosote may vary substantially as a function of working conditions related to temperature. Therefore, blood specimens were collected from separate groups during the winter, fall, and summer. Basal and induced CYP1A1 gene expression levels were not elevated in workers from any of the three seasonal studies. However, induced/basal (inducibility) CYP1A1 mRNA ratios from workers sampled in the summer (when actual exposures were greatest) were significantly higher when compared to those of controls (P < 0.01). These studies demonstrate the potential usefulness of specific gene expression assays in human peripheral lymphocytes for the assessment of carcinogen exposure in human populations.

Detection of cadmium exposure in rats by induction of lymphocyte metallothionein gene expression.

The induction of metallothionein (MT) gene expression in lymphocytes of rats was determined in order to detect exposure in vivo to cadmium. Both acute and chronic CdCl2 exposures resulted in the induction of the MT-1 gene in lymphocytes as measured by standard RNA Northern blot analysis. Twenty-four hours following an ip injection of 3.4 mg/kg CdCl2, a ninefold increase in MT gene expression was observed in lymphocytes, as well as five- and sevenfold increases in liver and kidney, respectively. Oral exposure of rats to 1-100 ppm CdCl2 via drinking water resulted in an approximate twofold enhanced MT signal in lymphocytes after 6 wk, and a threefold increase after 13 wk of exposure to 100 ppm Cd. No increases in lymphocyte MT gene expression were observed after 3 wk of Cd exposure. Liver MT gene expression was substantially induced following chronic Cd exposure, while kidney was not, although this organ had a higher basal expression of the MT-1 gene. Analysis of tissue Cd burdens demonstrated a dose-response Cd accumulation in liver and kidney, but only kidney burdens increased substantially with prolonged Cd exposure. These results demonstrate the utility of lymphocyte gene expression assays to detect in vivo toxicant exposure, and thus their applicability as molecular biomarker assays for human exposure assessment.

Ha-ras oncogene mutations in cell lines derived from rat tracheal implants exposed in vivo to 7,12-dimethylbenz[a]anthracene.

The frequency of Ha-ras mutations was determined as a function of neoplastic progression in cell lines derived from rat tracheal implants exposed in vivo to 7,12-dimethylbenz[a]anthracene. Restriction fragment-length polymorphism (RFLP) analysis revealed an A----T transversion in the second base of codon 61 in 2 of 11 cell lines. One of the positive cell lines was tumorigenic, but the other was neither tumorigenic nor anchorage independent, thus indicating a lack of correlation between neoplastic stage and ras mutation. Densitometry analysis of the RFLP bands indicated that approximately 50% of the cells within these two heterogeneous populations contained the mutation. Direct sequence analysis of polymerase chain reaction-amplified DNA confirmed these results and did not reveal any other mutations in this region of the Ha-ras gene.

The detection of DNA-protein complexes in vitro by an immunological assay.

We have developed an immunological assay to detect DNA-protein complexes (DPCs) in cell cultures treated with environmental toxicants. The assay uses an antiserum developed against K(2)CrO(4)-induced DPCs, which recognizes an acidic protein with a molecular weight of 95 kDaltons. The method uses a filter-binding assay to trap the DPCs from SDS-lysed cell cultures on polycarbonate filters, after which they are immunologically probed with the DPC antiserum. Cultures of Chinese hamster ovary cells were treated with K(2)CrO(4), formaldehyde or UV irradiation. DPCs were detected immunologically in cells treated with K(2)CrO(4) or UV irradiation, but not in those treated with formaldehyde. These results were similar to those obtained when DPCs were detected by filter-binding assay using radiolabelled cell cultures to measure the adherence of protein and DNA to the filters. In addition, both methods gave analogous dose responses of DPC formation in K(2)CrO(4)-treated cells. This novel immunological assay for detecting DNA lesions allows the rapid analysis of samples, which do not need to be radiolabelled, and thus it may have an application as a non-invasive test to screen for DNA-protein complexes.

An experimental model for oncogene activation during tumor progression in vivo.

Tumor progression usually involves a complex pattern of molecular alterations. In many human tumors oncogene amplification or activation has been associated with advanced stages of cancer. Transfection studies have demonstrated the ability of several cellular oncogenes to induce a more malignant phenotype in transformed cells. We have examined the role of c-myc in tumor progression in rat tracheal cell culture, and in rat skin tumors induced by ionizing radiation. In the latter in vivo model, c-myc amplification was found to occur as a function of tumor size. Serial biopsies of growing tumors confirmed the trend toward increased gene copy number with time and stage of progression. This effect was specific for the c-myc gene, in epithelial tumors. Evidence was found for a role of tumor heterogeneity and evolution of tumor cell subpopulations in determining the oncogene activation profile of individual tumors.

Oncogene expression in cell lines derived from rat tracheal implants exposed in vivo to 7,12-dimethylbenz[a]anthracene.

Expression of four oncogenes and two keratin genes was determined in rat tracheal epithelial cell lines derived from tracheal implants exposed in vivo to 7,12-dimethylbenz[a]anthracene. Cell lines were grouped into four stages of neoplastic progression based on phenotypic markers in order to correlate oncogene expression with stage of malignancy. Northern analysis of RNA revealed a significantly enhanced expression of the c-myc oncogene in the most tumorigenic or tumor-derived cell lines, whereas preneoplastic cells expressed approximately five-fold less transcript. Southern analysis of tracheal cell DNA did not demonstrate amplification of the c-myc gene in any of the positive cell lines. In contrast to c-myc, other oncogenes such as ras and fos were expressed in all cell lines, as well as in control cell cultures, to a similar extent. Patterns of differentiation were examined in these epithelial cell lines by determining the expression of two distinct keratin genes, KA-1 and KB-2. Both malignant and preneoplastic cells expressed the KB-2 gene at variably high levels, whereas the expression of the KA-1 keratin was barely detectable in any of the cell lines. The stage-specific expression of the c-myc oncogene in these tracheal cell lines suggests a correlation between the regulation of certain oncogenes and neoplastic progression in this model of respiratory carcinogenesis.

Benzo[a]pyrene- and formaldehyde-induced DNA damage and repair in rat tracheal epithelial cells.

The single and combined effects of 2 environmental carcinogens, benzo[a]pyrene (BAP) and formaldehyde (HCHO), on cell growth and DNA damage were assessed in a rat tracheal epithelial cell line, C18. Treatment of C18 cells with HCHO for 90 min reduced the calculated growth index at the highest concentration tested, 400 microM, while no growth effects were observed with BAP treatments. Combination treatments reduced the growth index to 75% of control values. Alkaline elution analysis of C18 cell DNA detected both DNA-protein crosslinks (DPC) and single-strand breaks (SSB) as a result of HCHO treatment, while BAP caused only SSB. HCHO-induced SSB were repaired within 2 h, whereas BAP-induced SSB persisted for at least 48 h. Combination treatment of cells with BAP followed by HCHO enhanced the number of SSB, but reduced DPC. The results are discussed in reference to earlier work which demonstrated the interaction in vivo between BAP and HCHO with respect to their carcinogenicity in rat tracheal implants.

Growth inhibition and DNA damage induced by benzo[a]pyrene and formaldehyde in primary cultures of rat tracheal epithelial cells.

The effects of benzo[a]pyrene (BAP) and formaldehyde (HCHO), alone and combined, on cell growth and DNA damage were determined in primary cultures of rat tracheal epithelial cells dissociated from rat tracheas. Cell cultures treated with 25 microM BAP for 24 h or 200 microM HCHO for 90 min did not have a marked reduction in cell growth. However, their combined treatment reduced cell growth by 60% of control when cultures were exposed to BAP followed by HCHO as well as the reverse order. None of these treatments significantly decreased cell viability as judged by dye exclusion, nor did they enhance cell terminal differentiation as measured by cornified envelope formation. Alkaline elution analysis of DNA damage detected both DNA-protein crosslinks (DPC) and DNA single-strand breaks (SSB) as a result of HCHO treatment, whereas BAP treatment caused only SSB. While HCHO-induced SSB were repaired within 2 h, BAP-induced SSB were detected 3 days after treatment. Combined treatment of cell cultures with BAP followed by HCHO resulted in more SSB than was obtained from either agent alone, but less DPC than was detected from HCHO alone. The increased number of SSB obtained from this combined treatment may be related to the marked enhancement of carcinogenesis observed in earlier in vivo-in vitro studies.

The detection of DNA-protein cross-links in rat tracheal implants exposed in vivo to benzo[a]pyrene and formaldehyde.

The DNA damage associated with benzo(a)pyrene (B[a]P) and formaldehyde (HCHO) exposure in rat tracheal implants was determined by alkaline filter elution adapted to measure DNA-protein cross-links (DPC) in vivo. In addition, histopathological responses of the tracheal epithelium were quantitated after multiple exposures to 20 micrograms B[a]P and 0.2% HCHO. Compared to either agent alone, combined exposure for 1-4 weeks caused an increase in cellular atypia and greater thickness of hyperplastic and metaplastic lesions. HCHO exposure resulted in a dose-dependent increase in DPC with a maximal response of 85% DNA filter retention at 0.2% HCHO, which were mostly removed by 72 h. B[a]P did not cause DPC, but when tracheas were pre-exposed to 20 micrograms B[a]P followed by 0.05% HCHO there was a 15% decrease in HCHO-induced DPC. This competition between B[a]P and HCHO for sites presumably on DNA does not offer a clear explanation for their markedly enhanced cocarcinogenicity observed in previous studies, but does demonstrate the interaction between the two agents in tracheal epithelium.

The induction of growth-altered cell populations (tumor-initiation sites) in rat tracheal implants exposed to benzo[a]pyrene and formaldehyde.

The effects of exposure to benzo[a]pyrene (BAP) and formaldehyde (HCHO), alone and combined, on the induction of carcinogenesis in rat tracheal implants was determined as the number of growth-altered cell populations (tumor-initiation sites, TIS) per trachea. While exposure twice-weekly for 4.5 months to 0.2% HCHO solution gave only a weak response (0.25 TIS/trachea), 2.37 TIS per trachea were detected after exposure to 20 micrograms BAP in the same regimen. The combination of BAP followed by HCHO had a greater response than either agent alone (7.83 TIS/trachea), while the reverse exposure gave 1.49 TIS per trachea, which was less than BAP alone. Thus, the induction of TIS by combined exposure to BAP and HCHO was dependent on the order of exposure, and could not be predicted from their individual exposures.

A model system for measuring comparative toxicities of cardiotoxic drugs with cultured rat heart myocytes, endothelial cells and fibroblasts. I. Emetine, chloroquine and metronidazole.

Neonatal rat hearts were separated into separate cultures of beating myocytes (M cells), endothelial cells (E cells) and fibroblasts (F cells). Their susceptibilities to the toxic effects of emetine, chloroquine and metronidazole were then compared using a quantitative metabolic inhibition test (QMIT) and morphologic and beating changes as indices of injury. Measurements on the same cultures were made at 6 h and 12 h daily for 7 days with E and F cells; with M cells for 3 days. Metronidazole was non-toxic for all cell types at 810 micrograms/ml, whether as the parent compound or after attempted rat liver microsomal activation. QMIT data, integrated as time-concentration effects, showed all cell responses to either emetine or chloroquine to be parallel (P less than 0.05), and their order of susceptibility to be: E greater than M greater than F cells. Although morphologic signs of injury and changes in beating are not readily evaluated statistically, there was a general parallelism between these indices of injury and those of QMIT. As judged by QMIT emetine was more toxic than chloroquine. However, at equivalent QMIT grades chloroquine produced greater effects on morphology and beating. Toxic concentration-50 values for chloroquine with the QMIT were similar to reported human toxic and lethal blood concentrations.

A model system for measuring comparative toxicities of cardiotoxic drugs for cultured rat heart myocytes, endothelial cells and fibroblasts. II. Doxorubicin, 5-fluorouracil and cyclophosphamide.

Separate cultures of beating myocytes (M cells), endothelial cells (E cells) and fibroblasts (F cells) from neonatal rat hearts were compared for their susceptibilities to the toxic effects of doxorubicin, 5-fluorouracil and cyclophosphamide. Toxic injury was measured as inhibition of metabolism and changes in morphology for all cell types, and changes in beating of myocytes. Cells were exposed to the anti-tumor agents for 3 days with myocytes and 7 days for endothelial cells and fibroblasts. All measures of injury were made several times the first day and daily thereafter, with the same cultures used throughout. Toxic effects of DOX and 5-FU were greater for E and F cells than for M cells, and all measures of toxicity were generally parallel. Cyclophosphamide, whether activated by liver microsomes or unactivated, was less toxic in general than the other agents, but it was more toxic for M cells than for E cells or F cells.

A quantitative metabolic inhibition test for screening toxic compounds with cultured cells.

The toxicity of substances for monolayer cultures of rat lung fibroblasts with phenol red as a pH indicator in the medium was measured by comparing the changes produced in medium color against 10 stable color standards. The toxicities of 6 concentrations each of actinomycin D, amphotericin B, chloroquine, 2-deoxyglucose, oligomycin, puromycin, and silicon dioxide were measured at 12 h and daily for 7 days on the same cultures throughout. Morphologic changes were monitored at the same times. The method, which simultaneously measures both concentration and time effects, was rapid, simple-to-perform, reproducible, low-in-cost, and quite sensitive. Its reproducibility depended on details of the cell culture methodology. The method is unsuitable for acidic or basic substances, or substances poorly-soluble in culture medium. Unless combined with morphologic evaluation, substances producing delayed toxicity may give misleading results.