Analysis of the candidate genes responsible for non-syndromic cleft lip and palate in Japanese people.

In order to assess the association of alleles for candidate genes with non-syndromic cleft lip and palate, DNA samples from 43 Japanese patients were compared with those from 73 control subjects with respect to the genes encoding transforming growth factor alpha (TGFalpha), TGFbeta and gamma-aminobutyric acid type A receptor beta3 (GABRB3). The restriction fragment length polymorphisms of the 3'-non-coding region of the TGFalpha gene K-primer region were observed after digestion with NcoI and HinfI. Allele 4 was the most common among cases of cleft lip with or without cleft palate, whereas allele 2 was the most common among controls. A significant difference was found in this region between groups with cleft lip (with or without cleft palate) and controls (chi2=10.190; P=0.017). Three alleles of the TGFbeta2 gene were tested, and allele 2 was the most common in both cases and controls. The proportion of allele 2 in the case group was greater than that in the control group, showing a significant difference between cases of cleft lip (with or without cleft palate) and controls (chi(2)=19.208; P<0.0001). No significant differences in variants of TGFbeta3 or GABRB3 between case and control populations were observed. Thus it is concluded that TGF genes play a role in craniofacial development, and that alleles of TGFalpha or/and TGFbeta2 are associated with cleft lip and cleft palate in Japanese populations.

Requirement of multiple DNA-protein interactions for inducible expression of RNR3 gene in Saccharomyces cerevisiae in response to DNA damage.

The RNR3 gene encodes the large subunit of ribonucleotide reductase. Transcription of this gene is induced 12-fold in response to DNA damage or by a DNA replication blocker. To investigate cis-acting regulation, deletion analysis of the promoter region of the RNR3 gene was performed and we identified two upstream-repressing sequences in the RNR3 regulatory region. An 18-base-pairs fragment, termed DNA-damage responsive element 1 (DRE1) located between -212 and -194 in this region was found to be essential for the induction of RNR3. This fragment contained a negatively acting sequence where a protein factor bound to the region during normal growth but disappeared by exposure to 4-nitroquinoline-1-oxide. The other repressive element homologue to DRE1 was located at -263 to -254. One possible upstream-activating sequence which regulates the basal expression of RNR3 was also found. These results show that at least three potential cis-elements are necessary for the inducible expression of yeast expression of yeast RNR3 in response to DNA damage.

Induction in the gene RNR3 in Saccharomyces cerevisiae upon exposure to different agents related to carcinogenesis.

The induction of the gene RNR3 was investigated in yeast Saccharomyces cerevisiae using RNR31 lacZ fusion. Gene induction was monitored by measuring beta-galactosidase activity. Various drugs that cause DNA damage effectively induced RNR3 expression; alkylating agents (cisplatin, mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine), a radical producer (bleomycin), and an intercalator (actinomycin D) induced RNR3. When yeast expressing rat CYP1A1 was exposed to 2-aminofluorene, a concentration-dependent induction of RNR3 was observed. Aflatoxin B1 also induced the expression of RNR3 in the same yeast strain concomitant with inhibition of cell growth. In control yeast, no induction of RNR3 was observed upon exposure to 2-aminofluorene or aflatoxin B1. Exposure to 2-acetylaminofluorene or benzo[a]pyrene did not lead to induction of RNR3 in yeast expressing CYP1A1. These results indicate that DNA damage by chemicals related to carcinogenesis induces RNR3, and that activation of these procarcinogens was required for DNA damage-dependent induction of RNR3.

Formation of 4,4'-methylene-bis(2-chloroaniline)-DNA adducts in yeast expressing recombinant cytochrome P450s.

N-Oxidation of 4,4'-methylene-bis(2-chloroaniline) (MBOCA) may lead to formation of DNA adducts. To determine if cytochrome P450s are involved in the formation of MBOCA derived-DNA adducts, yeast strains expressing rodent P450s were exposed to MBOCA, and 32P-postlabelling of nucleotides from yeast genomic DNA was done. Chromatographic analysis on PEI cellulose showed that, upon exposure to MBOCA for 1 h, nine DNA adducts were formed in yeast expressing phenobarbital-inducible rabbit P450 2B5. With a 4-h-exposure, all adducts increased in parallel. In cell-free experiments, the incubation of MBOCA with phenobarbital-induced rat microsomal fraction followed by incubation with thymus DNA, led to the formation of more than ten DNA adducts. When yeast expressing 3-methylcholanthrene-inducible rat P450 1A1 was exposed to MBOCA, one major and two minor adducts were formed. No adducts were detected in control yeast. These results show that recombinant rabbit P450 2B5 exhibits a potential activation of MBOCA and that rat P450 1A1 has some effect. The use of yeast expressing recombinant P450s and the technique of 32P-postlabelling facilitates a simple search for chemicals with carcinogenic potential.

Reversible lung lesions in rats due to short-term exposure to ultrafine cobalt particles.

Using an ultrasonic nebulizer, cobalt aerosols (MMAD = 0.76 microns, sigma g = 2.1) were generated from an aqueous suspension of ultrafine metallic cobalt particles (Uf-Co) with a primary diameter of 20 nm. Rats were exposed to Uf-Co aerosols at 2.72 +/- 0.44 mg/m3 for 5 hours (Exp. 1) or at 2.12 +/- 0.55 mg/m3 for 4 days at 5 hours/day (Exp. 2). Only minimum histopathological changes were observed in the lungs in Exp. 1. In Exp. 2, evidence of slight injury was noted, including focal hypertrophy or proliferation of the epithelium in the lower airways, damages of macrophages, intracellular edema of the type I alveolar epithelium, interstitial edema, and proliferation of the type II alveolar epithelium. A new finding in this study was the morphological transformation of some damaged type I cells to the juvenile form, which appeared to indicate the capability of self-repair of this cell type. The return to a juvenile form seemed to be a key response of type I cells during the early process of repair without cell division following non-lethal injury. Cobalt accumulated in the lungs after inhalation and was transferred rapidly to the blood. In conclusion, inhaled Uf-Co induced reversible pulmonary injury even after short-term exposure.