Mauro Pala

Sustained Activation of mTOR Pathway in Embryonic Neural Stem Cells Leads to Development of Tuberous Sclerosis Complex-Associated Lesions

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder characterized by hamartomatous neurological lesions that exhibit abnormal cell proliferation and differentiation. Hyperactivation of mTOR pathway by mutations in either the Tsc1 or Tsc2 gene underlies TSC pathogenesis, but involvement of specific neural cell populations in the formation of TSC-associated neurological lesions remains unclear. We deleted Tsc1 in Emx1-expressing embryonic telencephalic neural stem cells (NSCs) and found that mutant mice faithfully recapitulated TSC neuropathological lesions, such as cortical lamination defects and subependymal nodules (SENs). These alterations were caused by enhanced generation of SVZ neural progeny, followed by their premature differentiation and impaired maturation during both embryonic and postnatal development. Notably, mTORC1-dependent Akt inhibition and STAT3 activation were involved in the reduced self-renewal and earlier neuronal and astroglial differentiation of mutant NSCs. Thus, finely tuned mTOR activation in embryonic NSCs may be critical to prevent development of TSC-associated brain lesions.

Monitoring low benzene exposure : comparative evaluation of urinary biomarkers, influence of cigarette smoking and genetic polymorphisms

Benzene is a human carcinogen and an ubiquitous environmental pollutant. Identification of specific and sensitive biological markers is critical for the definition of exposure to low benzene level and the evaluation of the health risk posed by this exposure. This investigation compared urinary trans,trans-muconic acid (t,t-MA), S-phenylmercapturic acid, and benzene (U-benzene) as biomarkers to assess benzene exposure and evaluated the influence of smoking and the genetic polymorphisms CYP2E1 (RsaI and DraI) and NADPH quinone oxidoreductase-1 on these indices. Gas station attendants, urban policemen, bus drivers, and two groups of controls were studied (415 subjects). Median benzene exposure was 61, 22, 21, 9 and 6 μg/m3, respectively, with higher levels in workers than in controls. U-benzene, but not t,t-MA and S-phenylmercapturic acid, showed an exposure-related increase. All the biomarkers were strongly influenced by cigarette smoking, with values up to 8-fold higher in smokers compared with nonsmokers. Significant correlations of the biomarkers with each other and with urinary cotinine were found. A possible influence of genetic polymorphism of CYP2E1 (RsaI and/or DraI) on t,t-MA and U-benzene in subjects with a variant allele was found. Multiple linear regression analysis correlated the urinary markers with exposure, smoking status, and CYP2E1 (RsaI; R2 up to 0.55 for U-benzene). In conclusion, in the range of investigated benzene levels (<478 μg/m3 or <0.15 ppm), smoking may be regarded as the major source of benzene intake; among the study indices, U-benzene is the marker of choice for biomonitoring low-level occupational and environmental benzene exposure.

Occupational exposure to formaldehyde and biological monitoring of Research Institute workers

AIM The aim of this study was to verify the presence of a relationship between formaldehyde exposure in the work environment with biological markers of exposure and of effect. METHODS Exposure to formaldehyde (FA) of 36 workers in different laboratories of a Cancer Research Institute and biomarkers of exposure, such as formaldehyde human serum albumin conjugate (FA-HSA) and biomarkers of effect, such as chromosome aberration (CA), micronuclei (MN) and sister chromatid exchanges (SCEs) were measured in peripheral blood lymphocytes of the same workers. RESULTS Individual FA levels of exposure ranged from 4.9 microg/m(3) to 268.7 microg/m(3). Subjects with high FA exposure showed a significant increase of the biomarker of exposure FA-HSA, but biomarkers of effect did not show any significant differences. CONCLUSIONS A significant relationship was observed between occupational exposure to FA and a biological marker of exposure (FA-HSA). The markers of effect used (CA, MN and SCE) failed to indicate the presence of genetic damage.

Mutagenicity of polycyclic aromatic hydrocarbon fractions extracted from urban air particulates

Polycyclic aromatic hydrocarbon (PAH) fractions, purified from extracts of airborne particles collected in the area of Genoa municipality, were assayed for mutagenicity in the Salmonella/microsome test. PAH fractions accounted for only a portion of the total mutagenic activity and also displayed a different specificity of genetic activity, as compared to unfractionated material. The analysis of 224 samples collected from January 1986 to November 1987 in 10 different localities led to a large number of positive results in strain TA100 with S9 mix and, less frequently, also in TA98 without metabolic activation. Mutagenicity was related to the intensity of anthropogenic atmospheric pollution, and showed some seasonal variations, although it was not possible to discriminate particular sources of pollution on the basis of mutagenicity patterns. The mutagenic potency in TA100 (S9+) of airborne PAH fractions was significantly correlated with the concentration of individual PAHs in most of the monitored localities. The spectrum of mutagenicity of monthly samples pooled from several localities in S. typhimurium strains, with and without S9 mix, provided evidence for some contribution of nitro derivatives of PAHs or possibly also of other compounds present in the same fractions. The results obtained are discussed in view of their predictive value as indicators of potential health hazards, and of the reliability of this biological tool as a complement to chemical analyses in the evaluation of ambient air pollution.

Alkaline DNA fragmentation, DNA disentanglement evaluated viscosimetrically and sister chromatid exchanges, after treatment in vivo with nitrofurantoin

Nitrofurantoin was not positive as a carcinogen in long term assays. In vitro it was positive in some short term tests and negative in others. We have examined Nitrofurantoin for its capability of inducing DNA damage in vivo. With the alkaline elution technique, Nitrofurantoin appeared clearly positive in all the tissues examined (liver, kidney, lung, spleen and bone marrow). In the liver we also observed some cross-linking effect. In bone marrow cells Nitrofurantoin was also clearly positive in terms of sister chromatid exchanges (SCEs) induction. DNA damage in vivo was also examined with a viscosimetric method, more sensitive than alkaline elution. With this method the results were essentially negative, suggesting that the two methods detect different types of damage. In view of its positivity in many organs and in two short term tests in vivo, the carcinogenic potential of Nitrofurantoin should be reconsidered.

Assay of phenacetin genotoxicity using in vitro and in vivo test systems

Phenacetin was assayed in a battery of five short-term tests. (1) In a DNA-repair test using various Escherichia coli strains, the drug was not directly genotoxic nor did it induce nonreparable DNA damage in the presence of rat liver S9 fractions, while it was weakly active following activation with hamster liver S9. (2) In the Ames reversion test (strains TA97, TA98, TA100, and TA102 of Salmonella typhimurium, phenacetin reverted only TA100, and only in the presence of hamster liver S9. Mutagenicity was related to the concentration both of the drug and of the above metabolic system. There was no activation with hamster kidney S9, uninduced chicken liver S9, or with a variety of liver S9 preparations from rats treated with enzyme inducers (Aroclor 1254, phenobarbital, or 3-methylcholanthrene) and/or glutathione depletors (diethyl maleate or buthionine sulfoximine). Hamster liver S9 compared favorably to rat and even more to chicken liver S9 fractions also in activating various promutagens [3-amino-1-methyl-SH-pyrido (4,3-b)-indole, 2-aminofluorene, aflatoxin B1, benzo[a]pyrene, and benzo[a]pyrene-trans-7,8-diol] and in decreasing the mutagenicity of direct-acting compounds (4-nitroquinoline N-oxide and sodium dichromate). (3) Phenacetin was borderline positive in a forward mutation test (6-thioguanine resistance) in V79 cells, only in the presence of hamster liver S9, and gave negative results in the presence of rat liver S9 or without any metabolic system. (4) Following in vivo treatment, the alkaline elution assay did not reveal any DNA fragmentation in bone-marrow cells of ip-treated mice or in liver cells of rats treated by gavage. Apparent DNA damage was instead observed in the kidneys of rats receiving the drug by gavage or in the liver following ip administration. However, the effect was prevented (liver) or reduced (kidney) by preliminary perfusion of the organs, which discards (liver) or makes uncertain (kidney) the hypothesis of a true in vivo DNA damage. (5) Phenacetin ip induced in mouse bone-marrow cells a poor yet statistically significant increase in sister chromatid exchanges.

Sources and atmospheric concentrations of polycyclic aromatic hydrocarbons and heavy metals in two Italian towns (Genoa and La Spezia)

The same sampling and analytical methods were used to compare atmospheric pollution due to polycyclic aromatic hydrocarbons (PAHs) and heavy metals (Tl, Pb, Mn, Fe, Cr, V, Zr, Ni Cd) in two towns in Italy, Genoa and La Spezia, whose populations are 746, 785 and 112,602 respectively. Knowledge of the organic and inorganic composition of airborne particulates permits a reliable identification of the main sources of pollution which is required in order to identify populations at risk. In the urban area of Genoa and in La Spezia, traffic appears to provide a diffuse source of carcinogenic and toxic compounds in the atmosphere producing high and constant exposures to PAHs and lead along busy streets. In Genoa approximately 70,00 people (10% of residents) are considered to be exposed to the highest concentrations of toxic and cancerogenic pollutants emitted from this source. The highest daily PAH concentrations were found in the industrial areas; in Genoa, coke ovens were identified as the main localised sources of these compounds. According to meteorlogical and orographic characteristics for this area, for approximately 25,00 people (3% of the general population) may be exposed to pollutants emitted from this source over a maximum period equivalent to approximately 3 months each year. The highest individual doses of PAHs due to urban pollution inhaled by the population of Genoa and La Spezia were comparable to those produced by high exposure to passive smoke; the exposure to carcinogenic metals (Cr, NI, Cd) was relatively low. The mean concentrations of the analysed pollutants appeared to depend strictly on urban characteristics; no correlations were found with the size of the town.

Preliminary evaluation, using passive tubes, of carbon monoxide concentrations in outdoor and indoor air at street level shops in Genoa (Italy)

Abstract Preliminary information on carbon monoxide (CO) concentrations (exposure time: 8 h) both inside and outside 38 randomly selected shops situated on four heavy traffic streets of Genoa was obtained using passive diffusion tubes. Reproducibility and accuracy of this analytical method were tested in real outdoor urban conditions and found within 25%; the detection limit was 1 mgm−3 of CO. The highest mean CO concentrations (15.8 ± 2.2 mgm−3) were found inside shops on Balbi street, a narrow “canyon street”. Only in two small shops and two bars (both with many smokers) and in a delicatessen, were indoor CO concentrations significantly higher than outdoor values. The mean outdoor CO concentrations (mgm−3) along the four streets considered (XX Settembre, Balbi, Rolando, Fillak) were 7.4 ± 2.2; 14.5 ± 8.7; 5.8 ± 0.4; 10.5 ± 3.7, respectively. No statistical difference was found, comparing the mean indoor CO concentration with the mean CO outdoor value, measured simultaneously along the sidewalks of each street. CO concentrations in 10 shops without smokers and the nearest outdoor measurements were linearly correlated (r = 0.99; p

DNA damage in mouse and rat liver by caprolactam and benzoin, evaluated with three different methods.

Benzoin and caprolactam were examined for their capability of inducing alkaline DNA fragmentation in mouse and rat liver DNA after treatment in vivo. Three different methods were used. With the alkaline elution technique we measured an effect presumably related to the conformation of the DNA coil. With a viscometric and a fluorometric unwinding method we measured an effect presumably related to the number of unwinding points in DNA. For both compounds only the alkaline elution technique was clearly positive. The results suggest that both caprolactam and benzoin can induce an important change in the conformation of the DNA coil without inducing true breaks in DNA.

Effects of vitamin E on liver DNA

Vitamin E, both in the form of dl-alpha-tocopherol and dl-alpha-tocopheryl acetate, was capable of inducing an increased alkaline elution rate of liver DNA from rats treated i.p. with the vitamin. This activity was clearly both dose- and time-dependent. A statistically significant effect was observed at dosages (1.25-5.00 mg/kg) that are in the range of biological activity of the vitamin in the rat (reabsorption-gestation bioassay). Moreover, the effect was observed at dosages that are clearly not toxic. An increased alkaline elution rate of DNA is usually interpreted as suggestive of DNA damage, however recent observations seem to indicate that functional modifications of chromatin packaging can also affect the elution rate of DNA.