Biomedical subjects
M Paolini
Publications and source records attributed to M Paolini.
On the nature of non-genotoxic carcinogens. A unified theory including NGCs, co-carcinogens and promoters.
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On the hepatotoxicity of 1,1,2,2-tetrachloroethane.
Intoxication of male and female mice with a single dose (300 or 600 mg/kg) of 1,1,2,2-tetrachloroethane (TTCE) resulted in significant decreases in cytochrome P-450 (to 58-73% of the control) and NADPH-cytochrome (P-450) c-reductase (to 29-35% of the control) in hepatic microsomes. This was accompanied by an alteration of mixed function monooxygenases stemming from the marked reduction (to 20-64% of the control) of several oxidative activities to selected substrates towards different P-450 isozymes (classes IA1, IA2, IIB1, IIE1 and IIIA). As phase II markers, epoxide hydrolase (approximately 35% loss), UDP-glucuronosyl transferase (approximately 42% loss) and to a lesser extent glutathione S-transferase (approximately 17% loss) were all affected. Also, the activity of delta-aminolevulinic (ALA) synthetase was decreased (approximately 57% of the control). On the contrary, heme oxygenase activity was increased (up to 35%) at the maximal dose tested. The decrease of P-450-function may be explained in terms of an alteration in the rate of heme biosynthesis and degradation, provoking a loss of heme content (approximately 33%) as well as of the direct inactivation of both P-450 and reductase. Because of increasing evidence on the involvement of free radical intermediates in the case of toxicity of haloalkanes, electron spin resonance spectroscopy (ESR) spin-trapping in vivo techniques were used to characterize the possible free radical species involved in the observed liver damage. The results obtained with the spin-trap N-benzylidene-2-methylpropylamine N-oxide (phenyl t-butylnitrone, PBN) provide evidence for the formation and trapping of the CHCl2CHCl free radicals. The detection of conjugated diene signals by means of second-derivative spectrophotometry, have enabled us to show that in vivo lipid peroxidation may be one of the main mechanisms responsible for TTCE hepatotoxicity.
Strategies for advancement of short-term mutagenicity tests: on the optimal ionic strength for the liver microsomal assay.
The aim of this work was to optimize the ionic strength (tau) in the liver microsomal assay (LMA) in performing short-term genotoxicity tests. tau optimization would increase the sensitivity (i.e. decrease false negatives) and at the same time increase the specificity (decrease false positives). Such optimization depends upon the relative activities and stabilities of the liver polysubstrate cytochrome P450- and FAD-containing monooxygenase-dependent metabolizing enzymes present in the incubation mixtures. With regard to phase-I pathway, the expression of various P450-like activities (IA1, IA2, IIB1, IIE1, IIIA P450 classes) and thiobenzamide s-oxidase (as FAD-MFO marker), were examined in terms of their exact incubation conditions for the LMA during a period of preincubation (1 h) over the tau range 0.06-1.40. As a comparison with the phase-II pathway, the behaviour of glutathione S-transferases (total and pi class), glutathione S-epoxide transferase, epoxide hydrolase and UDP-glucuronosyl transferase were studied. Lipid peroxidation (LP) was also determined. Experiments were performed on S9 fractions derived from sodium phenobarbital, beta-naphthoflavone, isosafrol, ethanol and pregnenolone 16-alpha carbonitrile super-induced mouse liver. The maximal value of the mean specific activity (Asp), up to a 46% increase, was found at tau = 0.864 for oxidative reactions considered. On the contrary, a slight modulation of Asp for post-oxidative reactions was seen. LP was not changed appreciably by varying tau. In vitro DNA binding of the well-known premutagenic agent [14C]dimethylnitrosamine ([14C]DMNA), mediated by mouse hepatic microsomal enzymes, showed a significant increase of specific activity at tau = 0.864 (2.25-fold) compared to the usual tau (0.06) used. Additional confirmation of these results stems from mutagenesis experiments using DMNA on the diploid D7 strain of Saccharomyces cerevisiae as a biological test system. Indeed, a significant enhancement of mitotic gene conversion (up to 1.8-fold), mitotic crossing-over (2.6-fold) and reverse point mutation (2.6-fold) frequencies was achieved at tau = 0.86 compared to tau = 0.06 (traditional). These data show that tau = 0.86 can provide more convenient conditions for in vitro bioactivation (as exemplified by an increased Asp phase-I/Asp phase-II ratio), as well as DNA binding and genotoxic response.
Is clonal adaptation a product of evolution over the millennia?
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Expression and inducibility of drug-metabolizing enzymes in novel murine liver epithelial cell lines and their ability to activate procarcinogens.
Four novel nontransformed epithelial cell lines, isolated from fetal or adult mouse liver, were tested: (a) to determine the profile of xenobiotic metabolizing enzymes; (b) to evaluate the inducibility of the polysubstrate (cytochrome P-450-dependent) monooxygenase system by various classes of inducers; and (c) to assess the capacity of the cells to metabolize structurally different procarcinogens. With regard to the phase I pathway, the cells expressed various P-450 (class IA, IA2, IIB, IIE1, IIIA) and flavin adenine dinucleotide-containing monooxygenase-dependent bio-transformation enzyme activities at levels (in lines C2.8 and C6) comparable with those present in murine adult liver preparations. The expression of various P-450s was demonstrated also by immunoprecipitation assays using rabbit polyclonal antibodies. For the phase II pathway, cells expressed substantial levels of glutathione S-transferase, glutathione S-epoxide transferase, and UDP-glucuronosyltransferase. Low expression of epoxide hydrolase was observed. Induction of P-450 function by sodium phenobarbital, beta-naphthoflavone, isosafrole, ethanol, and pregnenolone 16 alpha-carbonitrile, monitored using specific P-450-linked activities, was considerably elevated (over 5-fold in class IIB with the C2.8 and C6 cell lines). The most competent C2.8 and C6 cell lines were able to activate benzo(a)pyrene, cyclophosphamide, dimethylnitrosamine, diethylstilbestrol, and 2-naphthylamine as shown by the significantly increased frequencies of mitotic gene conversion, mitotic crossing-over, and point [reverse] mutation in the diploid D7 strain of Saccharomyces cerevisiae after 4 [cyclophosphamide], 24 [benzo(a)pyrene,2-naphthylamine, dimethylnitrosamine] or 48 [diethylstilbestrol], h of exposure in the presence of 3 x 10(6) cells/flask. The degree of conservation and the inducibility of representative oxidative and postoxidative reactions in the novel epithelial cell lines C2.8 and C6, together with their ability to activate a wide spectrum of procarcinogens, offers a means to study the potential of chemicals for inducing DNA damage in short-term genotoxicity testing. In addition the cells may be suitable for analyzing the metabolic disposition of compounds and the multistage process of carcinogenesis.
Numerical simulation of thermal bone necrosis during cementation of femoral prostheses.
The implant of a femoral prosthesis is a critical process because of the relatively high temperature values reached at the bone/cement interface during the cementation of the infibulum. In fact, the cement is actually a polymer that polymerizes in situ generating heat. Moreover, the conversion of monomer into polymer is never 100%; this is dangerous because of the toxicity of the monomer. In this paper, we present a 3-D axisymmetric mathematical model capable of taking into account both the geometry of the implant and the chemical/physical properties of the cement. This model, together with its numerical simulation, thus represents a useful tool to set up the optimal conditions for the new materials developed in this orthopaedic field. The real complex geometry is assumed to be a bone/cement/metallic system having cylindrical symmetry, thus allowing the model to be reduced to two space variables. The cementation process is described by the Fourier heat equation coupled with a suitable polymerization kinetics. The numerical approximation is accomplished by semi-implicit finite differences in time and finite elements in space with numerical quadrature. The full discrete scheme amounts to solve linear positive definite symmetric systems preceded by an elementwise algebraic computation. We present various numerical simulations which confirm some critical aspects of this orthopaedic fixing technique such as thermal bone necrosis and the presence of unreacted residual monomer.
Pharmacokinetics of ursodeoxycholic acid in rat.
The pharmacokinetic behaviour and metabolism of ursodeoxycholic acid (UDCA) have been studied in the rat. After oral administration of both 3H-labelled (4 muCi/kg body wt) and unlabelled (20 mg) UDCA, UDCA appeared in serum almost entirely in conjugated form (taurine conjugated); UDCA was present in bile mostly as taurine conjugated; the more relevant metabolite is 3 alpha,6 alpha, 7 beta-trihydroxycholanoic acid which represents 10% of the total bile acid pool. UDCA increased bile flow and selectively decreased biliary cholesterol secretion, while phospholipid secretion was unaffected. Faecal UDCA excretion was 15-20% while the urinary extraction was 1.5% during 24 h. The data show that UDCA, when administered in high dose, is promptly secreted into bile almost entirely metabolized to tauroursodeoxycholic acid, where it (1) desaturates the cholesterol in bile, (2) exerts choleretic properties.
Mechanism of clastogenic and co-clastogenic activity of cremophore with benzene in mice.
Cremophore E1 (CR), a frequently used solubilizer and emulsifier in the pharmaceutical, cosmetic and animal-raising industries, is made up of ethylene oxide and castor oil. Since ethylene oxide has been shown to be a potent genotoxic agent, we have studied the clastogenic activity of CR and its co-clastogenic activity with benzene (BZ) in mice. Male CD1 mice were divided into untreated, vehicle control and experimental groups. Mice in the experimental groups were treated orally with 0.03, 0.3 or 3% CR in water, 440 mg/kg BZ in olive oil, BZ plus the three different doses of CR (1 h apart) or BZ plus 3% CR separated by 1, 3 and 5 h intervals. Mice were killed at 30 h after the treatment for the single-treatment groups and after the first treatment for the combined treatment groups. Bone marrow cells were harvested for determination of micronuclei (MN) frequencies in polychromatic erythrocytes (PCE). The presence of known genotoxic metabolites of benzene (phenol and trans,trans muconic acid) was quantitated in collected urine. The effect on hepatic cytochrome P450 isoenzyme expression in livers of treated mice were also analyzed. We found that CR did not induce any significant or dose-dependent increase in MN. However, CR enhanced the clastogenic activity of BZ in a dose-dependent manner (from 41.6 to 47.3, 60.5 and 67.1 MN/1000 PCE respectively; P less than 0.05). The combined treatment showed an inverse time-dependent change in MN frequencies when CR was administered at 1, 3 and 5 h after BZ (41.6 to 67.1, 43.4 and 42.0 MN/1000 PCE respectively). The enhancement effect of CR is apparently due to its ability to induce significantly the cytochrome P450I family when CR was administered 1 h after treatment with BZ. However, no positive synergistic effect was observed when the combined treatment intervals were extended to 3 and 5 h. Enhanced induction of these isoenzymes is correlated with increased metabolic activation of BZ to excrete increased amounts of trans,trans muconic acid, the putative active metabolite of BZ, in urine. Our integrated study demonstrates that an apparently innocuous agent that is consumed by the general population can enhance the genotoxic activity of a ubiquitous environmental carcinogen. The potential existence of this type of interaction in our daily lives is frequently overlooked and should be investigated.
Wide spectrum detection of precarcinogens in short-term bioassays by simultaneous superinduction of multiple forms of cytochrome P450 isoenzymes.
The use of Aroclor 1254 to induce S9 liver fractions is a standard method for conducting short-term genotoxicity assays. An alternative induction procedure, using beta-naphthoflavone (beta-NF), as a safe (non-carcinogenic) substitute for polychlorinated biphenyls, combined with sodium phenobarbital (PB), was found to be equally effective. The aim of this work is to realize a novel schedule of induction for the preparation of metabolizing systems containing a wider spectrum of induced cytochrome P450s. Five inducers of different 'classes' such as PB (class IIB P450s), beta-NF (IA), isosafrol (IA2), ethanol (IIE1) and pregnenolone 16 alpha-carbonitrile (IIIA) were injected daily both separately (to achieve maximal monooxygenase induction) in male and female mice. Induction was monitored using specific P450-linked activities. In the optimal schedule for complete induction, the various monooxygenases were greater (2- to 4-fold) than those achieved by the classical schedule. More than a 14-fold increase of total P450 and 3.3-fold increase of NADPH-cytochrome (P450) c-reductase activity, over those uninduced, account for the above increase. For example, there was a marked increase in the deethylation of ethoxyresorufin (37-fold) compared to the uninduced mice that was considerably higher than classical induction (8-fold over uninduced). On the contrary, phase II reactions i.e. epoxide hydrolase, glutathione S-transferase, glutathione S-epoxide transferase and UDP-glucuronosyl transferase, examined to compare the phase I/phase II ratios in the traditional and proposed procedures, were increased to a lesser extent (2-fold over uninduced). No significant sex differences were seen. Five precarcinogens specifically metabolized by each of the induced P450s elicited a higher mutagenicity response in the presence of superinduced fractions with respect to the classical one, when tested on Salmonella typhimurium (cyclophosphamide, benzo[alpha]pyrene, 2-naphthylamine and dimethylnitrosamine) or Saccharomyces cerevisiae D7 strain (diethylstilbestrol). These novel metabolizing biosystems, with an enhanced spectrum of induced P450s and oxidative/post-oxidative reaction rates, are recommended for detecting unknown xenobiotics in genotoxicity studies.
Dimethylsulfoxide as modifier of the organospecific mutagenicity of metronidazole in mice.
Mutagenicity and carcinogenicity of metronidazole (MT) are imputable to the formation of toxic intermediates, which include radical forms derived from the nitroreductive process. Since dimethylsulfoxide (DMSO), the "universal" solvent, can quench free radicals in vitro, it was suggested that DMSO might protect by scavenging free radical generation in vivo. This study wanted to evaluate if DMSO (given concomitantly or prophylactically) protects against the organospecific mutagenicity of MT in vivo by means of the intrasanguineous host-mediated assay. DMSO used as solvent showed a 20%-30% reduction in the mutation frequencies by MT. Prophylactic administration of DMSO for 3 d caused a suppression of the organospecific mutagenicity. However, some increases in the spontaneous mutation frequency and enhancement of MT mutagenicity in kidney were observed. The protective effect was paralleled by a decrease in NADPH cytochrome c (P450) reductase in liver, kidney, and to a lesser extent in lung microsomes from pretreated mice. Inhibition of mutagenic activity might be related to scavenging of radical species as supported by the lack of tissue specificity and no appreciable changes in specific enzyme activity. However, changes in reductase content in prophylactically pretreated mice can affect the quantitative biotransformation of MT to the proximal mutagen contributing to the observed suppression in mutation frequencies.
Strategies for optimization of short-term genotoxicity tests: the synergistic effect of NADPH and NADH on P450 function in processing pre-mutagens.
The synergistic effect of NADPH and NADH on P450 functions upon pre-mutagens requiring metabolism during the incubation conditions used in the liver microsomal assay (LMA) was studied. The mean specific activity (Asp) during 1 h of pre-incubation (LMA) of some microsomal mono-oxygenases (i.e. ethylmorphine N-demethylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase) examined with S9 fractions from sodium phenobarbital and beta-naphthoflavone pre-treated mice, was doubled when both NADPH and NADH were present. In contrast, when lipid peroxidation was used as the main enzymatic inactivation index, there was no appreciable change. In agreement with biochemical data, in vitro DNA binding of the pre-mutagenic agent [14C]-1,1,1,2-tetrachloroethane ([14C]TTCE), mediated by mouse hepatic enzymes, showed a significant enhancement (4.4-fold) of specific activity in the presence of both pyridine nucleotides. Mutagenesis experiments using TTCE in the diploid D7 strain of Saccharomyces cerevisiae (from stationary growth phase) as a biological test system, showed a significant enhancement of mitotic gene conversion and reverse point mutation frequencies when using NADPH plus NADH in the medium. Conversely, no positive results without NADH were seen. These findings lead us to suggest the routine use of both NADPH and NADH in order to increase the 'sensitivity' of in vitro mutagenicity screens.
Do cytochromes P-448 and P-450 have different functions?
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Tetrachloroethane, pentachloroethane, and hexachloroethane: genetic and biochemical studies.
Tetrachloroethane (TTCE), pentachloroethane (PCE), and hexachloroethane (HCE) were tested in diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension test with and without mammalian metabolic activation (S9). TTCE, PCE, and HCE gave positive results on cells harvested from logarithmic growth phase; only PCE induced a significant increase (P less than or equal to .01) of mitotic gene conversion and point reverse mutation on cells from stationary growth phase with metabolic activation (S9). The in vivo effects on cytochrome P450 content (cyt. P450), pentoxyresorufin O-dealkylase (P450-like, class IIB, PROD), and ethoxy-resorufin O-deethylase (P448-like, class IA, EROD) activities were examined in hepatic microsomes from mice 24 h after acute intoxication. All the halogenated hydrocarbons displayed a marked toxic effect as shown by the significant decrease in cyt. P450 levels (maximum of 76% decrease, with TTCE 753.2 mg/kg) and EROD (maximum of 69% decrease, with PCE 925.4 mg/kg), and to a lesser extent in PROD (maximum of 52.4% decrease, with HCE 3150 mg/kg). Although a general decrease of P450 functions was observed, the toxic effects of TTCE and PCE seem to be preferentially related to P448 forms.
Biliary excretion of mutagenic forms of nitroimidazoles in rats.
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[Critical aspects in determining total radioactivity of biological samples].
During measurements of radioactivity in some milk samples with liquid scintillation counter (about one year after the nuclear accident of Chernobyl) we have observed an increase of the values of scintillation fluid with the passing of time. Although this enhancement is absolutely small (about 2 c.p.m. in 500 min), it is very important for an exact measurement of samples at low counting, as those tested. Our protocol of measure provides for insertion of alternate blanks and samples in the automatic sample-holders of liquid scintillation counter. The values of measurement of samples are taken during the increase phase subtracting the value of blank interpolated on the increasing straight line from c.p.m. of sample. Finally, we report the collected values of the whole radioactivity in some milk samples: at least 5-6 nCi/L contrary to about 1 nCi/L of 137Cs reported by USL. In our opinion it is important to consider the whole radioactivity as measure of the overall biological danger of radioactive samples. In fact, this measurement takes into account also biologically very dangerous radionuclides as 3H, 14C, 90Sr.
The efficient preparation of corn oil solutions.
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EDTA affects cytochrome P450-dependent biotransformation reactions during incubations for the liver microsomal assay.
In order to optimize the condition of the liver microsomal assay (LMA), studies were carried out to determine the effects of EDTA on mixed-function oxidase activity and its stability under the exact incubation conditions for the LMA. Aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities as well as lipid peroxidation development (LP) in S9 liver fractions from beta-naphthoflavone and sodium phenobarbital (beta-NF + PB)- or Aroclor 1254 (AC)-treated mice were examined during a period of preincubation with EDTA ranging from 1 to 40 mM. At 5 mM EDTA, we obtained a strong inhibition of the microsomal LP as well as the greatest value of the mean specific activity (Asp) for both APD and pNAD activities. In agreement with the biochemical data, the presence of 5 mM EDTA in the incubation mixtures for the LMA significantly increased the mitotic gene conversion, mitotic crossing-over and point-reverse mutation of the well-known premutagen cyclophosphamide (30 mM) on the diploid D7 strain of Saccharomyces cerevisiae as the outcome of a greater metabolic activity. We concluded that the systematic use of 5 mM EDTA in LMA mixtures could improve the reliability and sensitivity of such a test.