Antonietta Martelli

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project.

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.

Update on genotoxicity and carcinogenicity testing of 472 marketed pharmaceuticals.

This survey is a compendium of genotoxicity and carcinogenicity information of 838 marketed drugs, whose expected clinical use is continuous for at least 6 months or intermittent over an extended period of time. Of these 838 drugs, 366 (43.7%) do not have retrievable genotoxicity or carcinogenicity data. The remaining 472 (56.3%) have at least one genotoxicity or carcinogenicity test result. Of the 449 drugs with at least one genotoxicity test result, 183 (40.8%) have at least one positive finding. Of the 338 drugs with at least one carcinogenicity test result, 160 (47.3%) have at least one positive result. Concerning the predictivity of genetic toxicology findings for long-term carcinogenesis assays, of the 315 drugs which have both genotoxicity and carcinogenicity data 116 (36.8%) are neither genotoxic nor carcinogenic, 50 (15.9%) are non-carcinogens which test positive in at least one genotoxicity assay, 75 (23.8%) are carcinogenic in at least one sex of mice or rats but test negative in genotoxicity assays, and 74 (23.5%) are both genotoxic and carcinogenic. Only 208 (24.8%) of the 838 drugs considered have all data required by current guidelines for testing of pharmaceuticals. However, it should be noted that a large fraction of the drugs considered were developed and marketed prior to the present regulatory climate. Although the laws do not require re-testing based on revised standards, in the absence of epidemiological studies excluding a carcinogenic risk to humans, a re-evalutation would be appropriate.

Ipriflavone inhibits osteoclast differentiation in parathyroid transplanted parietal bone of rats

SummaryIpriflavone, a synthetic isoflavone-derived flavonoid, was shown to have inhibitory effect on bone resorption. In order to study its mechanism of action directly on bone, 46 female Wistar rats were divided into six groups and medicated orally for 25 days as follows: groups 1 and 2 were given 1% carboxymethylcellulose solution (vehicle), groups 3, 4, 5, and 6 were administered ipriflavone at doses of 0.178, 0.356, 0.712, and 1.424 mmol/kg/day (suspended in vehicle), respectively. On the 22nd day, parathyroid glands, taken from donor rats, were transplanted in contact with the outer surface of the periosteum of both the right and the left parietal bones of rats from groups 2, 3, 4, 5, and 6. The group 1 rats underwent sham operation. Bone histomorphometry, performed on the ectocranial periosteum of parietal bones, showed that absolute erosion boundary, absolute eroded area, absolute erosion depth, number of tartrate-resistant acid phosphatase (TRAP)-positive polinucleated osteoclasts, and number of TRAP-positive mononucleated cells decreased in ipriflavone-treated rats compared with group 2 rats. The reduction was roughly proportional to the increase of drug dosage and reached statistical significance in rats of groups 5 and 6. The same parameters were extremely low in group 1 rats. Mineral apposition rate did not differ in any of the groups. Significant increase of serum calcium and significant decrease of serum phosphate were found in group 2 rats compared with group 1 rats, whereas no differences from controls were detected in ipriflavone-treated animals.The results demonstrate that ipriflavone has a direct inhibitory effect upon bone resorption, probably by reducing recruitment or differentiation of osteoclasts, rather than by inhibiting the resorption activity of differentiated osteoclasts. Ipriflavone also seems to exert a protective action against parathyroid hormone (PTH) diffusion from the site of parathyroid gland transplantation.

Genotoxic and carcinogenic effects of antipsychotics and antidepressants.

This review provides a compendium of the results of genotoxicity and carcinogenicity assays performed on marketed antipsychotics and antidepressants. Of the 104 drugs examined, 47 (45.2%) do not have retrievable data. The remaining 57 (54.8%) have at least one and often more than one genotoxicity and/or carcinogenicity test result. Of these 57 drugs, 33 (57.9%) have at least one positive finding: 24 tested positive in at least one genotoxicity assay, 14 in at least one carcinogenicity assay, and 5 gave a positive response in both. Concerning the predictivity of genetic toxicology findings for the result(s) of long-term carcinogenesis assays, 25 drugs have both genotoxicity and carcinogenicity data: 8 of them (32.0%) were neither genotoxic nor carcinogenic, 8 (32.0%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, 4 (16.0%) tested positive in at least one genotoxicity assay but were non-carcinogenic, and 5 (20.0%) gave a positive response in at least one genotoxicity assay and in at least one carcinogenicity assay. Only 16 (15.4%) of the 104 drugs examined have all data required by present guidelines for testing of pharmaceuticals, but it should be considered that a large fraction of them were developed and marketed prior to the present regulatory climate.

Are some progestins genotoxic liver carcinogens

Progestins (progestogens) are classified by the International Agency for Research on Cancer (IARC) as possibly carcinogenic to humans. In the last decade evidence has shown that a synthetic drug of this family, cyproterone acetate, is activated to a reactive species by the liver, and forms DNA adducts and elicits DNA repair in hepatocytes from both rats and humans. The response is similar in humans of both genders but markedly higher in female than in male rats. The promutagenic character of DNA lesions is indicated by the increase in liver of female rats of the frequency of micronucleated cells, of mutations, and of enzyme-altered preneoplastic foci. Two other synthetic progestins, chlormadinone acetate and megestrol acetate, and an aldosterone antagonist, potassium canrenoate, share with cyproterone acetate the 17-hydroxy-3-oxopregna-4,6-diene structure. While less extensively studied, results so far obtained indicate that they are capable of inducing genotoxic effects qualitatively similar to those of cyproterone acetate. The majority of progestins have not been systematically tested for genotoxicity and the generally negative responses obtained with the standard battery of genotoxicity tests might be the consequence of the use of inappropriate target cells and/or metabolic activation systems. Cyproterone acetate, is activated by the hepatocytes to reactive species of such short half-life that they react only with the DNA of the cell in which are formed. Therefore, it cannot be excluded a priori that other progestins will not display genotoxic effects when tested adequately. This hypothesis is supported by the knowledge that estrogen-progestin combinations used as oral contraceptives are classified by the IARC as carcinogenic to humans due to the increased risk of hepatocellular carcinoma. This risk should probably be ascribed to the progestin component, since estrogens are carcinogenic to humans due to the increased risk of endometrial and possibly of breast cancer but not liver cancer.

Genotoxicity and carcinogenicity studies of analgesics, anti-inflammatory drugs and antipyretics.

This survey is a compendium of genotoxicity and carcinogenicity information of analgesics, anti-inflammatory drugs and antipyretics. Data from 120 drugs were collected; 109 of them are still in the market. Of these 120 drugs, 58 (48.3%) do not have retrievable genotoxicity or carcinogenicity data. The remaining 62 (51.7%) have at least one genotoxicity or carcinogenicity test result. Of these 62 drugs, 31 have at least one positive finding: 26 tested positive in at least one genotoxicity assay, 13 in at least one carcinogenicity assay, and 8 gave a positive result in both at least one genotoxicity assay and at least one carcinogenicity assay. In terms of correlation between results of the various genotoxicity assays and absence of carcinogenic activity in mice and/or rats or in other species, 12 of 23 non-carcinogenic drugs tested positive in at least one of the various genotoxicity assay systems. Concerning the predictivity of genetic toxicology findings for the result(s) of long-term carcinogenesis assays, 35 drugs have both genotoxicity and carcinogenicity data: 11 of them (31.4%) were neither genotoxic nor carcinogenic, 4 (11.4%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, 12 (34.3%) tested positive in at least one genotoxicity assay but were non-carcinogenic, and 8 (22,9%) gave a positive response in at least one genotoxicity assay and in at least one carcinogenicity assay. Only 22 (18.3%) of the 120 drugs considered have all data required by current guidelines for testing of pharmaceuticals, but a large fraction of them were developed and marketed prior the present regulatory climate.

Species, sex and inter-individual differences in DNA repair induced by nine sex steroids in primary cultures of rat and human hepatocytes.

Sex steroids, due to the generally negative responses observed in routinely employed standard genotoxicity assays, are considered epigenetic carcinogens. Some doubts on this conviction are raised by the results of recent studies providing evidence that cyproterone acetate and two structural analogues, chlormadinone acetate and megestrol acetate, are genotoxic in female rats but only for the liver, and in primary human hepatocytes from donors of both genders. The experimental evidence suggests that the metabolic activation of these molecules to reactive species and the consequent formation of DNA adducts occur only in the intact hepatocyte. Since the possibility that other sex steroids cause a liver-specific genotoxic effect cannot be ruled out a priori, we investigated nine drugs of this family for their ability to induce DNA repair synthesis in primary cultures of rat and human hepatocytes. Each steroid was tested in cultures from at least two male and two female donors of each species. Hepatocytes were exposed for 20h to sub-toxic concentrations ranging from 1 to 50 micro M, and DNA repair induction was measured by quantitative autoradiography. In primary rat hepatocytes, induction of DNA repair indicative of a frankly positive response was detected in cultures from: 2/2 males and 3/3 females with drospirenone, 2/2 males and 1/2 females with ethinylestradiol, 1/2 males and 1/2 females with oxymetholone, 1/2 males with norethisterone, 1/4 females with progesterone, and 1/4 males with methyltestosterone. Consistent negative responses were obtained with testosterone and stanozolol. A few inconclusive responses were observed in rat hepatocytes exposed to progesterone, medroxyprogesterone, norethisterone, methyltestosterone and oxymetholone. In contrast, under the same experimental conditions the nine sex steroids provided frankly negative responses in the large majority of cultures of primary hepatocytes from both male and female human donors; the only exceptions being the inconclusive responses obtained in cultures from two of the donors exposed to norethisterone and to ethinylestradiol, and from one of the donors exposed to testosterone, methyltestosterone, and stanozolol. These results and previous findings concerning cyproterone and its structural analogues suggest that sex steroids differ for their ability to induce DNA repair, and that their genotoxicity may be: (i) different in rat and human hepatocytes, (ii) dependent on the sex of the donor, and (iii) affected by inter-individual variability.

Evaluation of omeprazole genotoxicity in a battery of in vitro and in vivo assays

Omeprazole, a proton pump inhibitor of wide use in the treatment of gastric acid-related disorders, was evaluated for its genotoxic effects in both rat and human cultured cells and in the intact rat. DNA repair synthesis, as revealed by autoradiography, was detected in primary cultures of metabolically competent rat hepatocytes exposed to concentrations ranging from 10 to 100 mg/l, but the responses cannot be considered as clearly positive. Under the same experimental conditions any significant evidence of DNA repair was absent in primary hepatocytes from two human donors. At the same concentrations a modest but dose-related increase of micronucleated cells, that reached the level of statistical significance at 33 mg/l, was present in primary rat hepatocytes and in one of two human donors. In human lymphocytes exposed to subtoxic concentrations ranging from 0.78 to 12.5 mg/l a reproducible concentration dependent clastogenic effect was absent. In partially hepatectomized female rats treated with a single p.o. dose of 1000 mg/kg, the frequency of micronucleated cells was 5.2-fold higher than in controls in the liver, but only 2.0-fold higher in polychromatic erythrocytes of the bone marrow. In rats of the same sex given azoxymethane as initiator of colon carcinogenesis the oral administration for 8 successive weeks of 10 mg/kg omeprazole on alternate days increased the response to azoxymethane, as indicated by the occurrence in colon mucosa of a modest but statistically significant increase in both the average number and size of aberrant crypt foci. Taken as a whole, our results suggest that omeprazole behaves as a weak genotoxic agent for the rat liver. Reliable information about the potential genotoxic risk to humans requires further studies on primary cells from a wide number of donors.

Is lipid peroxidation associated with DNA damage

Specific binding to DNA of lipid peroxidation products was studied in rat hepatocytes labeled with [14C(U)]arachidonic acid after incubation at 37 degrees C either in the absence or in the presence of 200 microM FeSO4. The results obtained show that: (1) production of malondialdehyde-like thiobarbituric-reactive substances occurred in the absence of FeSO4 and was increased, albeit quite variably, by exposure to this pro-oxidant; (2) a low but appreciable binding of radioactivity to DNA and protein was constantly detected in 5 independent experiments; (3) there was no quantitative correlation between malondialdehyde formation and the amount of DNA-bound and protein-bound radioactivity, and any meaningful evidence of a GSH-depletion effect was absent. Taking into account the possible biosynthetic incorporation of radioactivity into DNA, the results of this study must be interpreted with caution, and solely as indicating that in the intact cell the covalent binding to DNA of reactive species generated by lipid peroxidation, if it occurs, should be minimal, corresponding in our experimental conditions approximately to 0.01 mumole of radioactive arachidonic acid per mole nucleotides.

Genotoxicity testing of chloramphenicol in rodent and human cells

The results of this work, carried out to extend the limited information at present available on the genotoxic potential of chloramphenicol (CAP), indicate that in millimolar concentrations this antibacterial agent produced a minimal amount of DNA fragmentation in both V79 cells and metabolically competent rat hepatocytes. Moreover, a level of DNA-repair synthesis indicative of a weak but positive response was detected in primary cultures of liver cells obtained from 2 of 3 human donors, and a borderline degree of repair was present in those prepared from rats. The promutagenic character of CAP-induced DNA lesions was confirmed by a low but significant increase in the frequency of 6-thioguanine-resistant clones of V79 cells, which, however, was absent when the exposure was done in the presence of co-cultured rat hepatocytes. Finally, oral administration to rats of 1/2 LD50 CAP did not increase the incidence of either micronucleated polychromatic erythrocytes or micronucleated hepatocytes. Taken as a whole these findings suggest that CAP should be considered a compound intrinsically capable of producing a very weak genotoxic effect, but only at concentrations about 25 times higher than those occurring in patients treated with maximal therapeutic dosages.