Greg Cosma

Relationship between genotype and function of the human CYP1A1 gene

A comparative study of human CYP1A1 genotypes and enzymatic activity was performed in a racially diverse population in order to determine frequencies of CYP1A1 genetic polymorphisms and the relationship between CYP1A1 genotype and function. Restriction fragment length polymorphism analyses revealed significantly higher frequencies of a variant Msp1 polymorphism in Asians versus European-Americans, while African-American CYP1A1 genotypic frequencies more closely approximated those of Asians. Comparison of CYP1A1 genotypes at the Msp1 locus to a polymorphic site in exon 7 of the gene revealed a higher frequency of variant genotypes at the Msp1 site. Measurement of lymphocyte CYP1A1 enzyme activity by the ethoxyresorufin O-deethylase assay revealed significantly elevated levels of inducible enzyme activity among variant exon 7 genotypes when compared to wild-type genotypic individuals. These results demonstrate racially distinct patterns of CYP1A1 genotypes, and suggest a functional link between genotype and catalytic activity of the cytochrome P-450 protein responsible for the metabolism of many carcinogenic polycyclic aromatic hydrocarbons.

Relationship between ambient air pollution and DNA damage in Polish mothers and newborns.

Industrialized regions in Poland are characterized by high ambient pollution, including polycyclic aromatic hydrocarbons (PAHs) from coal burning for industry and home heating. In experimental bioassays, certain PAHs are transplacental carcinogens and developmental toxicants. Biologic markers can facilitate evaluation of effects of environmental PAHs on the developing infant. We measured the amount of PAHs bound to DNA (PAH-DNA adducts) in maternal and umbilical white blood cells. The cohort consisted of 70 mothers and newborns from Krakow, Poland, an industrialized city with elevated air pollution. Modulation of adduct levels by genotypes previously linked to risk of lung cancer, specifically glutathione S-transferase MI (GSTM1) and cytochrome P4501A1 (CYP1A1) Msp restriction fragment length polymorphism (RFLP), was also investigated. There was a dose-related increase in maternal and newborn adduct levels with ambient pollution at the womens place of residence among subjects who were not employed away from home (p < or = 0.05). Maternal smoking (active and passive) significantly increased maternal (p < or = 0.01) but not newborn adduct levels. Neither CYP1A1 Msp nor GSTM1 polymorphisms was associated with maternal adducts. However, adducts were significantly higher in newborns heterozygous or homozygous for the CYP1A1 Msp RFLP compared to newborns without the RFLP (p = 0.04). Results indicate that PAH-induced DNA damage in mothers and newborns is increased by ambient air pollution. In the fetus, this damage appears to be enhanced by the CYP1A1 Mspl polymorphism. ImagesFigure 1

Rat lung metallothionein and heme oxygenase gene expression following ozone and zinc oxide exposure

We have conducted exposures in rats to determine pulmonary responses following inhalation of two common components of welding fumes, zinc oxide and ozone. To examine their effects on target-inducible gene expression, we measured mRNA levels of two metal-responsive genes, metallothionein (MT) and heme oxygenase (HO), in lung tissue by RNA slot-blot analysis. A 3-hr exposure to ZnO fume via a combustion furnace caused a substantial elevation in lung MT mRNA at all concentrations tested. Exposures to 5 and 2.5 mg/m3 ZnO resulted in peak 8-fold increases in MT mRNA levels (compared to air-exposed control animal values) immediately after exposure, while 1 mg/m3 ZnO exposure caused a 3.5-fold elevation in MT mRNA. These levels returned to approximate control gene expression values 24 hr after exposure. In addition, ZnO exposure caused an immediate elevation in lung HO gene expression levels, with 8-, 11-, and 5-fold increases observed after the same ZnO exposure levels (p < 0.05). Like MT gene induction, HO mRNA values returned to approximate control levels 24 hr after exposure. In striking contrast to the induction of MT and HO gene expression after ZnO exposures, there was no elevation in gene expression following a 6-hr exposure to 0.5 and 1 ppm ozone, even when lungs were examined as late as 72 hr after exposure. Our results demonstrate the induction of target gene expression following the inhalation of ZnO at concentrations equal to, and below, the current recommended threshold limit value of 5 mg/m3 ZnO. Furthermore, the lack of effect of ozone exposure on MT and HO gene expression suggests no involvement of these genes in the acute respiratory response to this oxidant compound.

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.

Role of H-ras in the malignant progression of rat tracheal epithelial cells.

The effect of an activated H-ras oncogene on the progression of neoplasia was studied in transformed rat tracheal epithelial cells. Nude mouse tumours produced by injection of these cells exhibited a higher fraction of cells containing the mutantras gene than did the injected cells, while a subclone that lacked theras mutation was much less tumorigenic than parental cells. Serial passage of one cell line containing aras mutation resulted in an increase in the fraction ofras-mutated cells, which suggests that, in this line,ras activation may confer a selective advantagein vitro as well. However, this was not seen in anotherras-containing line, suggesting the importance of alternative pathways in malignant progression of rat tracheal epithelial cells.

EFFECTS OF MUTATIONALLY ACTIVATED HA-RAS ON C-FOS EXPRESSION KINETICS IN RAT TRACHEAL EPITHELIAL CELLS

The rat tracheal implant model was used to characterize the role of activated Ha‐ras in the neoplastic progression of heterogeneous rat tracheal epithelial (RTE) cell populations. An activated Ha‐ras‐containing cell line, RTE 2‐2, and its subclone, RTE 2‐2n, which possesses only Ha‐ras proto‐oncogene alleles, were studied to determine whether activated ras could interact with the downstream signal transduction targets fos and myc and alter their cell‐cycle‐dependent expression in vitro. Transformed RTE cell lines with activated Ha‐ras displayed earlier fos expression, with a peak at 15 min after serum stimulation. These cell lines also displayed a more accelerated loss of fos mRNA than seen in cells without activated Ha‐ras. The effects on fos expression kinetics were seen only in cell lines with activated ras and were not related to the transformed phenotype of the cells. No change in myc expression kinetics were observed in any RTE cell line. These results suggest that mutations in ras can lead to alterations in nuclear components of the ras signaling pathway at the level of gene transcription.