Francesco Giorgelli

In vitro assessment of salvage pathways for pyrimidine bases in rat liver and brain

In this paper we extend our previous observation on the mobilization of the ribose moiety from guanosine to xanthine catalyzed by rat liver extracts (Giorgelli et al., Biochim. Biophys. Acta 1335 (1997) 16-22). The data show that in rat liver and brain extracts the activated ribose, stemming from inosine and guanosine phosphorolysis as ribose 1-phosphate, can be used to salvage uracil to uracil nucleotides. Uridine is an intermediate. The salvage process occurs even in the presence of excess inorganic phosphate suggesting that uridine phosphorylase may function in vivo as an anabolic enzyme. Ribose 5-phosphate cannot substitute for inosine, guanosine or ribose 1-phosphate as ribose donor. When inorganic phosphate was substituted with arsenate, hindering the formation of ribose 1-phosphate, no ribose transfer could be observed. A similar pathway occurs at the deoxy level. The deoxyribose moiety of deoxyinosine can be used to salvage thymine to thymine nucleotides, again in the presence of excess inorganic phosphate. Our results introduce a novel aspect of the salvage pathway, in which ribose 1-phosphate seems to play a pivotal role.

Recycling of α-d-ribose 1-phosphate for nucleoside interconversion

Mobilization of the ribose moiety and of the amino group of guanosine may be realized in rat liver extract by the concerted action of purine nucleoside phosphorylase and guanase. Ribose 1-phosphate formed from guanosine through the action of purine nucleoside phosphorylase acts as ribose donor in the synthesis of xanthosine catalyzed by the same enzyme. The presence of guanase, which irreversibly converts guanine to xanthine, affects the overall process of guanosine transformation. As a result of this purine pathway, guanosine is converted into xanthosine, thus overcoming the lack of guanosine deaminase in mammals. Furthermore, in rat liver extract the activated ribose moiety stemming from the catabolism of purine nucleosides can be transferred to uracil and, in the presence of ATP, used for the synthesis of pyrimidine nucleotides; therefore, purine nucleosides can act as ribose donors for the salvage of pyrimidine bases.

Deoxyadenosine metabolism in a human colon-carcinoma cell line (LoVo) in relation to its cytotoxic effect in combination with deoxycoformycin

We have assessed the intracellular metabolism of 2′‐deoxyadenosine in a human colon‐carcinoma cell line (LoVo), both in the absence and in the presence of deoxycoformycin, the powerful inhibitor of adenosine deaminase. The combination of 2′‐deoxyadenosine and deoxycoformycin has been reported to inhibit the growth of LoVo cells in culture. In this paper we demonstrate that the observed toxic effect is strictly dependent on cell density. In the absence of deoxycoformycin, 2′‐deoxyadenosine is primarily deaminated to 2′‐deoxyinosine and then converted into hypoxanthine. In the presence of the inhibitor, the deoxynucleoside, in addition to a phosphorylation process, undergoes phosphorolytic cleavage giving rise to adenine. The conversion of 2′‐deoxyadenosine to adenine might represent a protective device, emerging when the activity of adenosine deaminase is reduced or inhibited. There is much evidence to indicate that the enzyme catalyzing this process may be distinct from methylthioadenosine phosphorylase and S‐adenosyl homocysteine hydrolase, which are the enzymes reported to be responsible for the formation of adenine from 2′‐deoxyadenosine in mammals. Int. J. Cancer 75:713–720, 1998.© 1998 Wiley‐Liss, Inc.

Correlation between mutagenicity of airborne particles and air pollution parameters in eleven Italian towns

Organic extracts from airborne particles collected in 11 Italian towns between February and April, 1988, were tested for mutagenicity on TA98 and TA100 (± S9), and their nitroreductase (NR) deficient Salmonella strains, by the use of the Ames plate incorporation assay. Mutagenic responses were fitted by an equation which takes into account toxic effects on tester organisms. Generally parallel responses were obtained with the two Salmonella strains, but the TA98 gave, mostly, higher increases of revertants over the control level. No dramatic decreases in mutagenicity were observed with the NR derivative strains, except in a few cases with TA98NR and, more frequently, with TA100NR strains. During air sampling, temperature, atmospheric pressure, light, wind strength and direction, SO2, CO, NO2, O3 and non-methanic hydrocarbons (NMHC) concentrations were continuously monitored. Meteorological variables seem not to be significantly correlated with mutagenicity variations, while the highest correlation (r = 0.91) was observed between induced reversion in TA98 (+ S9) and NMHC concentration in air. Therefore, in spite of the wide range of different types of towns included in the study, air NMHC concentration can be considered a good predictor for the mutagenicity of the total organic material extracted from particles of urban air.

Genetic Toxicology of Airborne Particulate Matter Using Cytogenetic Assays and Microbial Mutagenicity Assays

The mutagenicity of airborne particulate matter was assayed using four different tests, namely, the Ames Salmonella test, sister chromatid exchanges (SCEs), chromosomal aberrations (CAs), and the micronucleus (MN) test in human lymphocytes. Two types of extracts were used for all the tests. The first was a sample obtained from urban air in winter which showed high mutagenic activity. The second was a summer sample, again from urban air, which showed very low activity. The particulate matter was assayed as a whole organic extract, and in the form of different fractions. In the first sample, we observed that SCE and CA tests were more sensitive than the Ames test. Since both tests in eukaryotic cells are time-consuming and expensive, we tried to assess the value of the MN test with a second sample showing low activity. Again, the three cytogenetic tests were more sensitive than the test on Salmonella, and in particular the MN test seems to be a useful test in conjunction with the Ames test. As a first screening, it could substitute for the other two tests.

Role of the phosphorolysis of deoxyadenosine in the cytotoxic effect of the combination of deoxyadenosine and deoxycoformycin on a human colon carcinoma cell line (LoVo)

In LoVo cells, phosphorolytic activity acting on deoxyadenosine plays a major role in the resistance to the cytotoxic effect of the combination of deoxynucleoside with deoxycoformycin. In fact, the observed dependence of toxicity on cell density appears to be related to the metabolic conversion of deoxyadenosine into adenine. The phosphorylation of the deoxynucleoside, which represents the first step towards the formation of the cytotoxic agent dATP, proceeds at a significantly lower rate as compared to the phosphorolysis of deoxyadenosine. The analysis of the levels of deoxyadenosine and its derivatives in the incubation media reveals that the rates of disappearance of deoxyadenosine and of formation of adenine increase in concert with the reduction of the effect on cell survival. J. Cell. Biochem. 80:241–247, 2000.

6-Thioguanine resistance in a human colon carcinoma cell line with unaltered levels of hypoxanthine guanine phosphoribosyltransferase activity

Cell populations resistant to high doses (30 μM) of 6‐thioguanine (6‐TG, 6‐TGr cells) were selected from a human colon carcinoma cell line, LoVo. This cell line, which lacks hMSH2, a component of the human mismatch binding heterodimer hMutSα, is resistant to low doses of 6‐TG. The level of activity of hypoxanthine‐guanine phosphoribosyltransferase, the enzyme responsible for the phosphoribosylation of the thiopurine, was comparable to that expressed in the parental cells. No significant difference was found in the levels of enzyme activities involved in the conversion of 6‐TG or its derivatives into non‐toxic compounds. In contrast, a significant difference was found in the uptake kinetics of 6‐TG in the 2 cell types. Net uptake of 6‐TG ceased after 100‐sec incubation in the 6‐TGr cells, while it appeared to continue throughout the 10‐min incubation in the wild‐type cells. As a consequence, after 10‐min incubation, the total amount of 6‐TG taken up by the parental LoVo cells was approximately 3 times higher than that present in the 6‐TGr cells. Int. J. Cancer 82:556–561, 1999.