Increased frequency of wild-type arylamine-N-acetyltransferase allele NAT2*4 homozygotes in Portuguese patients with colorectal cancer

Here we report that colorectal cancer patients show a markedly higher frequency (3-fold) of wild-type NAT2*4 allele homozygotes than the control population. However, a marked difference in NAT2*4/NAT2*4 genotype frequency associated with the patients gender was observed pointing to a male-specific effect of this genotype as a risk factor in colon cancer. The arylamine-N-acetyltransferase (E.C. 2.3.1.5.) NAT2, a phase II detoxification enzyme, has been implicated in procarcinogen activation, namely from food contained arylamines, cigarette smoking, as well as environmental amines of various types. NAT2 is encoded by a polymorphic gene presenting several allelic variants encoding partially inactive enzymes expressed in human liver and colon. Epidemiological studies based on phenotype determination have long indicated the importance of the NAT2 active phenotype as a susceptibility factor in colorectal cancer. In the present study we investigated the NAT2 allelic frequencies and genotype distribution in a group of 114 unrelated colorectal cancer patients, in parallel with 201 healthy Portuguese subjects. We first demonstrate that the frequency of the wild-type NAT2*4 allele in the Portuguese sample population (23.4%) does not significantly differ from the values described for other Europeans. Besides the 3-fold higher frequency of NAT2*4 homozygotes found in colorectal cancer subjects, the NAT2*4/NAT2*5A compound genotype, known to determine a faster acetylator phenotype than other heterozygotic combinations, also increased by the same order of magnitude. These two genotypes represent 32% of the patients population versus 11% of the healthy controls. Taken together, our results strongly indicate that NAT2 genotype, particularly NAT2*4 allele zygosity, constitutes an individual susceptibility trait associated with sporadic colorectal cancer development, probably due to the local dietary habits in Portugal.      

Basic fibroblast growth factor prolongs the proliferation of rat cortical progenitor cells in vitro without altering their cell cycle parameters

Basic fibroblast growth factor (bFGF) has been shown to influence the survival, proliferation and differentiation of a variety of cell types in the nervous system. In this investigation we have examined the action of bFGF on: (i) the rate of proliferation; (ii) cell cycle parameters; (iii) the maintenance of cell division; (iv) the recruitment of quiescent cells; and (v) the degree of differentiation of cortical progenitor cells in cultures prepared from E16 rat embryos. The proliferation rate (labelling index) of cortical progenitor cells doubled in the presence of bFGF over 48 h. However, the lengths of the cell cycle phases were unchanged. Clones marked with a recombinant retrovirus on the first day in vitro (DIV) grew significantly larger in the presence of bFGF. Furthermore, many of the clones examined in control cultures had ceased to divide after a maximum of four cell cycles, whereas almost all clonally related cells were still dividing in the presence of bFGF 4 days later, i.e. for at least six cell cycles. Basic FGF also stimulated the division of quiescent progenitor cells, which otherwise would have differentiated or undergone cell death. The degree of neuronal and glial differentiation was studied after 5 DIV using MAP-2 and GFAP immunocytochemistry. In the presence of bFGF, the percentage of MAP-2-labelled cells was less than half that of control cultures, whereas the number of cells immunoreactive for nestin (a marker of progenitor cells) remained very high. Cells immunoreactive for GFAP were present in bFGF-treated cultures, yet were extremely rare in control conditions. These experiments show that bFGF, a potent mitogen for cortical progenitor cells, has no effects on the parameters of their cell cycle but extends their proliferative capability, promotes their survival and delays their differentiation into neurons.      

Clinical relevance of P-glycoprotein expression in de novo acute myeloid leukemia

Cytofluorimetric detection of the multidrug resistance (MDR)-associated membrane protein (P-170) was performed at the time of diagnosis in 158 patients with acute myeloid leukemia using the C219 monoclonal antibody (MoAb). In 108 of these cases the JSB1 MoAb was also tested. An improved histogram subtraction analysis, based on curve fitting and statistical test was applied to distinguish antigen-positive from antigen-negative cells. A marker was considered positive when more than 20% of the cells were stained. At onset, P-170 was detected in 43% of cases with C219 and in 73% of cases with JSB1. There was a strict correlation between C219 and JSB1 positivity, as all C219+ cases were also positive for JSB1 MoAb (P < .001). No relationship was found between sex, age, organomegaly, and MDR phenotype. Significant correlation was found between CD7 and both C219 and JSB1 expression (P < .001 and .001, respectively). C219-negative phenotype was more often associated with a normal karyotype (24 of 55 with P = .030). Rhodamine 123 (Rh123) staining and flow cytometry analysis showed a significantly decreased mean fluorescence in 51 C219+ and 38 JSB1+ patients compared to 42 MDR negative ones (P < .001). The rate of first complete remission (CR) differed both between C219+ and C219- cases and between JSB+ and JSB- ones (30.9% v 71.1% and 35.4% v 93.1%, respectively, P < .001). Of the 21 C219+ patients who had yielded a first CR, 19 (90.4%) relapsed, compared with 28 of 64 (43.7%) C219- patients (P < .001). Of the 28 JSB1+ patients in first CR, 17 (60.7%) relapsed relative to 8 (29.6%) of 27 JSBI- ones (P = .021). A higher rate of relapses among MDR+ compared with MDR- patients was observed both for C219 and JSB1 MoAbs taken separately (C219 80% v 44%; JSB1 52% v 27%), with no relationship to age. The survival rates (Kaplan-Meyer method) were significantly shorter both in C219+ patients and in JSB1+ cases (P < .001). Disease-free survival curves followed this same trend. The combination (C219- JSB1+) identified a subset of patients with an intermediate outcome compared to C219 positive cases. The prognostic value of both markers (C219 and JSB1) was confirmed in multivariate analysis. These results suggest that the assessment of MDR phenotype by flow cytometry may be an important predictor of treatment outcome.     

Contribution of hydrazines-derived alkyl radicals to cytotoxicity and transformation induced in normal c-myc-overexpressing mouse fibroblasts

Several hydrazine derivatives (HD) tested so far have pharmacological activities, but many also have toxic side effects, including carcinogenesis. Their toxicity has been ascribed to carbocations (via formation of azoxy intermediates), alkyl radicals or reactive oxygen species. Cytotoxicity and transformation by carbocations is widely accepted, but the role of alkyl radicals is still questioned. We have investigated the cytotoxicity of HD to mouse fibroblasts in three activation systems in which enhanced alkyl radical formation is demonstrated by electron spin resonance/spin-trapping. Cytotoxicity was assayed by inhibition of [3H-methyl]thymidine uptake into DNA of Balb/c 3T3 and/or Myc 9E fibroblasts (normal Balb/c 3T3 cells over-expressing the c-myc proto-oncogene). Based on the results obtained in the cytotoxicity assays we also investigated the transforming potential of procarbazine (PCZ) and methylhydrazine (MeH) activated by horseradish peroxidase (HRP) using the Myc 9E cell line, which aims at the activation of a second cooperating oncogene. Our results show that: (i) cytotoxicity of HD to mouse fibroblasts is increased by HRP activation of MeH, phenelzine and PCZ, which displayed enhanced alkyl radical formation, but not of 1,2-dimethylhydrazine (DMH), which did not produce increased alkyl radical formation under these conditions; (ii) cytotoxicity of neutrophil-activated MeH (producing a 10-fold higher concentration of methyl radicals), is more pronounced than DMH; (iii) MeH and DMH activated by prolonged auto-oxidation in 24-h incubations have comparable cytotoxicity and alkyl radical formation; and (iv) PCZ and MeH activation by HRP to alkyl radicals increased the transformation induced in Myc 9E cells. Taken together, our results strongly support a role for hydrazine-derived alkyl radicals in HD- induced cytotoxicity and cell transformation.      

Dental erosions and gastro-oesophageal reflux disease in institutionalized intellectually disabled individuals

OBJECTIVE: Both exogenous acids, from the diet, and endogenous acids, from stomach juice, can dissolve the enamel mineral, resulting in dental erosionS. Gastric acid may reach the mouth by gastro-oesophageal reflux disease (GERD), recurrent vomiting, rumination and regurgitation. These conditions are frequently found in the intellectually disabled population. Therefore, we investigated the presence of dental erosions in combination with GERD among intellectually disabled inhabitants, with an IQ<50, taken from three Dutch institutes.
MATERIALS AND METHODS: At random 63 individuals underwent an oesophageal pH test and dental screening and possible predisposing and attributable factors were determined. An abnormal pH level was defined as a pH <4, >4.5% of the measured time. Subjects with dental erosions were compared to those without dental erosions.
RESULTS: In 29 out of 63 (46.0%) cases evidence of dental erosions was found. In 19 of these 29 subjects with erosions (65.5%) GERD was diagnosed, compared to nine (26.5%) out of 34 subjects without erosions ( P = 0.04). In the subjects with erosions mean duration of pH <4 was 15.6% (range: 0.5–90.5) compared to 6.3% (range 0–40.4) in subjects without erosions ( P = 0.02).An IQ <35 was found to be predisposing ( P <0.001).
CONCLUSION: In this population of 63 institutionalised intellectually disabled persons dental erosions were diagnosed in 46%.Sixty-five per cent of them had GERD. Individuals with longer duration of pH <4 than 6.3% of the measured time and with an IQ < 35 were at higher risk to develop dental erosions. This study shows that dental erosions in the intellectually disabled population might be an oral manifestation of GERD.     

Genotoxicity of propoxur and its N-nitroso derivative in mammalian cells

N-Nitroso propoxur (NP) can be synthesized from a widely used N- methylcarbamate insecticide, propoxur, in vitro in the laboratory. Because of the extensive use of aerosol propoxur, the adverse effect on cells of respiratory origin is worth elucidating. In this report, two mammalian cell cultures from respiratory tissues [a hamster lung fibroblast, V79, and a primary rat tracheal epithelial cell (RTE)], were used to investigate the genotoxicity of propoxur and NP. NP was more cytotoxic than propoxur, with LC50s (20 and six times smaller, respectively in V79 and RTE cells. NP significantly induced sister chromatid exchange (> or = 0.01 microg/ml), chromosome aberration (> or = 2.5 microg/ml) and hprt gene mutation (> or = 0.5 microg/ml) in V79 cells, and cell transformation (> or = 0.2 microg/ml) in RTE cells. Results of chromosome aberration and hprt gene mutation indicated that the major pre-mutagenic lesion induced by NP must be the O6- methylguanine adduct, which frequently mispairs with thymine and thus gives rise to a GC-->AT transition. Propoxur was not mutagenic to either type of cells. However, it inhibited gap-junctional intercellular communication in V79 cells, which indicates that propoxur could act through some epigenetic mechanisms, such as tumor promotion or cell proliferation, in the multiple process of chemical carcinogenesis.