Ciprofloxacin: in vivo genotoxicity studies

The fluoroquinolone ciprofloxacin is widely used in antimicrobial therapy. It inhibits the bacterial gyrase and in high concentrations in vitro also the functionally related eukaryotic topoisomerase-II, which resulted in genotoxic effects in several in vitro tests. In order to evaluate the relevance of these findings, ciprofloxacin was tested in vivo for genotoxic activity using the following test systems: micronucleus test in bone marrow of mice, cytogenetic chromosome analysis in Chinese hamster, dominant lethal assay in male mice and UDS tests in primary rat and mouse hepatocytes in vivo. These results are compared with already published in vitro and in vivo studies with ciprofloxacin. All in vivo genotoxicity revealed no genotoxic effect for ciprofloxacin. In addition, ciprofloxacin was found to be non-carcinogenic in two rodent long-term bioassays. Therefore, ciprofloxacin is considered to be safe for therapeutic use.     

In vivo studies on genotoxicity of pure and commercial linuron

The ureic herbicide linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea] (CAS 330-55-2) was investigated for genotoxicity in a series of in vivo experiments. Since human exposure to herbicides is not only to the active principles, but also to all the chemicals present in the commercial formulation, we tested both pure and commercial linuron. Groups of rats were treated with gavage containing different doses of the herbicide (pure compound or commercial formulation) for 14 days. The doses were 150, 300 and 450 mg/kg b.wt. for the pure compound and 315.8, 631.6 and 947.4 mg/kg b.wt. for the commercial formulation (47.5% of linuron). Faeces and urine were collected at regular intervals. Urine specimens were analysed for their mutagenic metabolites, thioethers and d-glucaric acid content. Faeces extracts were tested for mutagenicity. Linuron's ability to cause DNA damage and cytogenetic effects was also investigated after treating groups of rats once with different doses of pure or commercial linuron. DNA single-strand breaks were assessed in rat liver using the alkaline elution technique and the single-cell microgel electrophoresis assay (SCGE: `comet' assay), and in rat testes cells with the SCGE assay. Micronuclei induction was analysed in rat bone marrow erythrocytes. Results obtained were mainly negative when the excretion of mutagenic metabolites in urine and faeces of animals treated with the pure compound or with the linuron-based commercial formulation were monitored, whereas an increase in the urinary excretion of thioethers and d-glucaric acid was observed in rats treated with the commercial formulation. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the treated animals. However, linuron affected the viability of hepatocytes isolated from animals treated with higher doses. This cytotoxicity was accompanied by the induction of DNA single-strand breaks in the liver, as seen by the alkaline elution assay. The potential of pure linuron to induce in vivo DNA damage was confirmed with the microgel electrophoresis technique (`comet' assay). Cytotoxicity was also seen in rat testes cells. However, no indication of DNA damage was visible.     

Comparative in vivo and in vitro genotoxicity studies of airborne particle extract in mice

The genotoxicity of an acetone extract of locally collected airborne particles was evaluated both in vitro and in vivo using the sister-chromatid exchange (SCE) assay in mice. At the highest concentration (5.36 mg/5 ml culture), the extract caused approximately a 3-fold increase in SCEs over controls in mouse bone marrow and spleen primary cells in vitro. However, the same airborne particle extract did not induce a significant increase in the SCE level over controls in vivo in mouse bone marrow and spleen cells when administered intraperitoneally or through oral gavage. This indicates that bone marrow and spleen primary cell cultures can be used in in vitro genotoxicity studies of complex mixtures, and that the genotoxicity of airborne particles detected in the in vitro system cannot always be detected in vivo with the same cell types. In addition, the same acetone extract of airborne particles caused dose-related his+ revertants in the strain TA98 of Salmonella typhimurium, both with and without S9 activation. The significant finding of this study is that the in vitro genotoxicity results of airborne particle extract may not be very meaningful in an in vivo situation.     

Macleaya cordata extract and Sangrovit genotoxicity. Assessment in vivo

Background: Sanguinarine (SG) has been reported to form DNA adducts in vitro and increase the levels of DNA single strand breaks in the blood and bone marrow of mice treated intraperitoneally with SG. Recently, we showed no genotoxic effects of orally administrated 120 mg/kg feed Macleaya cordata extract (a mixture of sanguinarine and chelerythrine) in pigs or rats in 90-day studies. The goal of this paper was to assess the possible genotoxicity of M. cordata extract when included as a dietary admixture to rodents at concentrations providing 600 mg/kg feed and 100, 7000 or 14000 mg/kg feed Sangrovit (natural feed additive containing M. cordata extract and powdered M. cordata) in a 90-day pilot study. Methods and Results: The rats consumed ad libitum either the standard diet or the diets containing 367 ppm of sanguinarine and chelerythrine in M. cordata extract, and 5, 330, or 660 ppm of total alkaloids in Sangrovit for 90 days. The DNA adducts formation in liver was analyzed by (32)P-postlabeling technique and DNA single strand breaks in lymphocytes were evaluated by Comet assay. The results showed that M. cordata extract and/or Sangrovit induced no DNA damage to rat lymphocytes or hepatocytes after 90-days oral administration. Conclusions: Data from the studies described in this paper and the fact that Sangrovit given to the rats in our experiments were higher than the recommended dose (50 to 100 mg/kg feed), argue strongly in favour of the use of Sangrovit in live stock.     

Ciprofloxacin: mammalian DNA topoisomerase type II poison in vivo

Ciprofloxacin (CF), a fluoroquinolone widely used as a potent antimicrobial drug, was evaluated in vivo in mouse bone marrow cells for its ability to induce clastogenicity and DNA damage in terms of increased sister-chromatid exchange (SCE) frequencies. Doses of 0.6, 6 and 20 mg/kg body weight of CF given intraperitoneally induced a positive dose-dependent significant clastogenicity (trend test α ⩽ 0.05), though the effects were not specific for specific phases of the cell cycle.The DNA-damaging effect observed as increased SCE frequencies using doses of 0.15, 0.30, 0.60, 1.2 and 6 mg/kg body weight showed a significant dose-dependent increase (trend test α ⩽ 0.05; lowest effective concentration 1.2 mg/kg of body weight).Compared to a potent eukaryotic DNA topoisomerase type II poison, etoposide (VP-16, 0.5, 1 and 5 mg/kg body weight, given intraperitoneally), ciprofloxacin produced comparable dose-dependent SCE frequency increases. Ciprofloxacin was postulated to be specific for the target DNA gyrase, the prokaryotic homologue of DNA topoisomerase type II enzyme. The present paper along with the existing earlier data strongly suggest that topoisomerase type II and DNA gyrase are physiological targets for the drug action. In view of the present significant in vivo mammalian DNA topoisomerase type II-mediated genotoxicity and clastogenicity data, ciprofloxacin should be administered with caution.     

In vivo genotoxicity evaluation of dimethylarsinic acid in MutaMouse.

Dimethylarsinic acid (DMA) induces DNA damage in the lung by formation of various peroxyl radical species. The present study was conducted to evaluate whether arsenite or its metabolite, DMA, could initiate carcinogenesis via mutagenic DNA lesions in vivo that can be attributed to oxidative damage. A transgenic mouse model, MutaMouse, was used in this study and mutations in the lacZ transgene and in the endogenous cII gene were assessed. When DMA was intraperitoneally injected into MutaMice at a dose of 10.6 mg/kg per day for 5 consecutive days, it caused only a weak increase in the mutant frequency (MF) of the lacZ gene in the lung, which was at most 1.3-fold higher than in the untreated control animals. DMA did not appreciably raise the MF in the bladder or bone marrow. Further analysis of the cII gene in the lung, the organ in which DMA induced the DNA damage, revealed only a marginal increase in the MF. Following DMA administration, no change in the cII mutation spectra was observed, except for a slight increase in the G:C to T:A transversion. Administration of arsenic trioxide (arsenite) at a dose of 7.6 mg/kg per day did not result in any increase in the MF of the lacZ gene in the lung, kidney, bone marrow, or bladder. Micronucleus formation was also evaluated in peripheral blood reticulocytes (RETs). The assay for micronuclei gave marginally positive results with arsenite, but not with DMA. These results suggest that the mutagenicity of DMA and arsenite might be too low to be detected in the MutaMouse.     

The in vivo and in vitro genotoxicity of aromatic amines in relationship to the genotoxicity of benzidine.

Benzidine and 12 related aromatic amines have been studied for the effects of substituent groups and pi orbital conjugation on their genotoxicity as measured by their mutagenicity in vitro with Salmonella and by chromosomal aberrations (CA) in vivo in the bone-marrow cells of mice. The in vitro studies indicated increases in mutagenicity with increases in the electron withdrawing ability of para' substituents. Mutagenicity also increases with increased conjugation as shown by the degree of planarity of the biphenyl compounds and by comparing the mutagenicities of biphenyl amines to stilbenes as well as to ethylene bridged diphenyl compounds. The relative in vitro mutagenicity results were not predictive of relative in vivo CA results. The 3 most genotoxic compounds in vivo were the conjugated amines without substituents in the para' position. The CA values for 4-aminostilbene were exceptionally high. These in vivo results indicate increased genotoxicity for benzidine analogs without substitution in the para' position.     

Mechanism of genotoxicity of diethylstilbestrol in vivo

Diethylstilbestrol (DES) is a carcinogen in humans and rodents which has eluded mechanistic clarification of its carcinogenic action. In vitro and in vivo, binding of DES to DNA has been found previously, but covalent DNA adducts could not be identified. In this study, the nature of binding was investigated by 32P-postlabeling, a rapid and highly sensitive assay for covalent DNA damage, to distinguish between a genotoxic or epigenetic mechanism of carcinogenesis by DES. A unique and distinct DNA adduct pattern was observed in kidney, liver, uterus (or testes) of female (or male, respectively) Syrian hamsters treated with a single injection of DES (200 mg/kg body weight). This set of DNA adducts closely matched patterns generated in vitro by reaction of diethylstilbestrol-4',4'-quinone with DNA or 2'-deoxyguanosine 3'-monophosphate. The major and several minor DES-DNA adducts in vivo had identical chromatographic mobilities in 11 different solvent systems with corresponding adducts obtained in vitro. The major adduct spot, generated in vitro by reaction of diethylstilbestrol-4',4'-quinone and DNA, was chemically unstable (half-life at 37 degrees C: 4-5 days). The persistence in vivo of these DNA modifications was low (biological half-life: 14 h) presumably because of chemical instability in concert with DNA repair. After injection of identical dosages of DES, adduct concentrations were 4-6-fold higher in females than in males. These results demonstrate that DES is capable of covalently modifying DNA. Moreover, diethylstilbestrol-4',4"-quinone is the major reactive metabolic intermediate responsible for the genotoxic activity of DES. Tumors are expected to arise only in rapidly dividing cells due to the short biological lifetimes of DES-DNA adducts.     

UKEMS genotoxicity trial 1981: In vivo assay systems

4CMB was tested in 7 mouse micronucleus assays (3 i.p., 2 oral, 1 s.c., 1 foetal), 2 mouse sperm-head morphology assays (1 i.p., 1 s.c.), 1 dominant lethal test in mouse (oral), 3 SLRL tests in Drosophila (2 feeding, 1 injection) and one somatic cell segregation assay in Drosophila (feeding).4HMB was tested in 4 mouse micronucleus assays (2 i.p., 1 oral, 1 s.c.), 2 mouse sperm morphology assays (1 i.p. and 1 s.c.), 1 SLRL test and 1 somatic cell segregation assay in Drosophila (both feeding).BC was tested in 4 mouse micronucleus assays (2 i.p., 2 oral, 1 s.c.), 2 sperm-morphology assays (1 i.p., 1 s.c.), 2 SLRL tests and 1 somatic cell segregation assay in Drosophila (all feeding).All the mouse assays gave negative results with all 3 compounds. The response of 4CMB and BC was not reproducible when tested under similar conditions in the SLRL test in Drosophila. All 3 compounds gave a positive response in the somatic cell segregation assay in Drosophila.     

Reinvestigation of in vivo genotoxicity studies in man. I. No induction of DNA strand breaks in peripheral lymphocytes after metronidazole therapy

Although a rodent carcinogen, metronidazole is widely used in humans for the treatment of infections with anaerobic organisms. Metronidazole is mutagenic for microorganisms, but has a mainly negative data base for mammals and humans. Therefore, metronidazole is generally considered as a non-genotoxic carcinogen. Only the results of two human in vivo studies would allow the classification of metronidazole as genotoxic carcinogen: (1) the induction of DNA strand breaks; and (2) the induction of chromosome aberrations in peripheral lymphocytes after metronidazole therapy. Because the classification of metronidazole as genotoxic carcinogen would imply enormous consequences with respect to its application, both studies were reinvestigated very thoroughly. The present report describes the reinvestigation of the induction of DNA strand breaks after metronidazole therapy. Each two probes of lymphocytes of metronidazole-treated patients (3×500 to 3×750 mg/day for 5–8 days) were examined separately for the appearance of DNA strand breaks before and after treatment. In total, 400 nuclei were examined per patient. Immediately before the first, and 30 min to 2 h after the last application, 2×10 ml blood per patient was sampled, transported to the laboratory at 15–20°C to make DNA repair more difficult, and examined within the next 4–7 h for DNA strand breaks. At the same time, the individual metronidazole blood plasma levels were measured. In contrast to the published reports, no induction of DNA strand breaks after metronidazole therapy could be observed in the present study. As the applied doses (15 750 mg vs. 4800 mg) and the plasma level (up to 25 μg/ml vs. not measured) of metronidazole were much higher than in the published study, the relevance of the clearly negative result is obvious. As induction of DNA strand breaks is a frequent prerequisite for genotoxicity, metronidazole should be considered as a non-genotoxic carcinogen, and not as a genotoxic carcinogen.     

An in vivo assay for small intestine genotoxicity

The induction of nuclear aberrations (NA) (apoptotic bodies and micronuclei) in duodenal crypts in a dose-dependent manner was associated with administration of agents known to induce tumours in the small intestine. These included X-irradiation, N-methyl-N-nitrosourea, (MNU), benzo[a]pyrene (B[a]P), and 1,2-dimethylhydrazine (DMH), which were found to induce NA in cells in the proliferative region of crypts 24 h after they were given to mice. Methylurea (MU) and benzo[e]pyrene (B[e]P), which are non-carcinogenic structural analogues of MNU and B[a]P, respectively, did not induce NA under similar conditions. Based on these results, the ability of an agent to induce NA in the small intestine appears to reflect of its oncogenic potential in that organ.     

In vitro and in vivo genotoxicity of radiofrequency fields

There has been growing concern about the possibility of adverse health effects resulting from exposure to radiofrequency radiations (RFR), such as those emitted by wireless communication devices. Since the introduction of mobile phones many studies have been conducted regarding alleged health effects but there is still some uncertainty and no definitive conclusions have been reached so far. Although thermal effects are well understood they are not of great concern as they are unlikely to result from the typical low-level RFR exposures. Concern rests essentially with the possibility that RFR-exposure may induce non-thermal and/or long-term health effects such as an increased cancer risk. Consequently, possible genetic effects have often been studied but with mixed results. In this paper we review the data on alleged RFR-induced genetic effects from in vitro and in vivo investigations as well as from human cytogenetic biomonitoring surveys. Attention is also paid to combined exposures of RFR with chemical or physical agents. Again, however, no entirely consistent picture emerges. Many of the positive studies may well be due to thermal exposures, but a few studies suggest that biological effects can be seen at low levels of exposure. Overall, however, the evidence for low-level genotoxic effects is very weak.     

Evaluation of the potential in vivo genotoxicity of quercetin

Quercetin, a naturally occurring flavonol commonly detected in apples, cranberries, blueberries, and onions, has been reported to possess antioxidant, anti-carcinogenic, anti-inflammatory, and cardioprotective properties. While positive results have been consistently reported in numerous in vitro mutagenicity and genotoxicity assays of quercetin, tested in vivo, quercetin has generally produced negative results in such studies. Furthermore, no evidence of carcinogenicity related to the oral administration of quercetin was observed in chronic rodent assays. In order to further define the in vivo genotoxic potential of quercetin, a bone marrow micronucleus assay and an unscheduled DNA synthesis (UDS) assay were conducted in Wistar rats. Administered orally to male rats at dose levels of up to 2000 mg/kg body weight, quercetin did not increase the number of micronucleated polychromatic erythrocytes (MN-PCE) 24 or 48 h following dosing in the micronucleus assay. Likewise, orally administered quercetin (up to 2000 mg/kg body weight) did not induce UDS in hepatocytes of male or female rats. While measurable levels of metabolized quercetin were observed in rat plasma samples for up to 48 h after dosing, peaking at 1 h following treatment administration, the unmetabolized aglycone was not identified in either plasma or bone marrow. With the exception of only a few rats, the aglycone was also not detected in liver tissue. These results demonstrate that quercetin is not genotoxic under the conditions of these assays and further support the negative results of previously conducted in vivo assays.     

Evaluation of the potential in vivo genotoxicity of quercetin

Quercetin, a naturally occurring flavonol commonly detected in apples, cranberries, blueberries, and onions, has been reported to possess antioxidant, anti-carcinogenic, anti-inflammatory, and cardioprotective properties. While positive results have been consistently reported in numerous in vitro mutagenicity and genotoxicity assays of quercetin, tested in vivo, quercetin has generally produced negative results in such studies. Furthermore, no evidence of carcinogenicity related to the oral administration of quercetin was observed in chronic rodent assays. In order to further define the in vivo genotoxic potential of quercetin, a bone marrow micronucleus assay and an unscheduled DNA synthesis (UDS) assay were conducted in Wistar rats. Administered orally to male rats at dose levels of up to 2000 mg/kg body weight, quercetin did not increase the number of micronucleated polychromatic erythrocytes (MN-PCE) 24 or 48 h following dosing in the micronucleus assay. Likewise, orally administered quercetin (up to 2000 mg/kg body weight) did not induce UDS in hepatocytes of male or female rats. While measurable levels of metabolized quercetin were observed in rat plasma samples for up to 48 h after dosing, peaking at 1h following treatment administration, the unmetabolized aglycone was not identified in either plasma or bone marrow. With the exception of only a few rats, the aglycone was also not detected in liver tissue. These results demonstrate that quercetin is not genotoxic under the conditions of these assays and further support the negative results of previously conducted in vivo assays.     

Cycloheximide genotoxicity in in vitro and in vivo test systems

The aim of this study was to investigate if there was any genotoxic effect produced by the antibiotic cycloheximide, widely used as a fungicide in agriculture as well as in everyday laboratory practice. The battery of test systems included the bacterium Salmonella typhimurium (strains TA98 and TA100), the yeast Saccharomyces cerevisiae (D7), Allium cepa somatic cells and mouse bone marrow cells. This combination of test systems enabled us to establish possible effects caused by cycloheximide at different levels of the genome and to indicate a possible mechanism of action. The results obtained in experiments showed that cycloheximide did not induce frameshift or base-pair substitution mutations in S. typhimurium regardless of metabolic activation. In S. cerevisiae cycloheximide had only toxic effects but no increase of mitotic gene conversion was noticed under the conditions of the experiment. However, in A. cepa somatic cells as well as in mouse bone marrow cells cycloheximide showed its activity causing different genetic damages, e.g., chromosome breaks, mitotic disturbances and nuclear abnormalities.     

Mutagenicity and genotoxicity of isatin in mammalian cells in vivo

Isatin (1H-indole-2,3-dione) is a synthetically versatile substrate used for the synthesis of heterocyclic compounds and as a raw material for drug synthesis. Isatin and its derivatives demonstrate anticonvulsant, antibacterial, antifungal, antiviral, and anticancer properties. We evaluated the genotoxic and mutagenic effects of acute (24h) and repeated (14d) exposure to isatin in vivo, using the comet assay and the micronucleus test. Three doses (50, 100, and 150mg/kgb.w.) were administered to mice via gavage. Doses were selected according to the LD(50) of isatin, estimated in a preliminary test to be 1g/kgb.w. To evaluate the results, parametric (ANOVA/Tukey) and non-parametric (Kruskal-Wallis/Dunn's post hoc test) tests were used, according to the nature of the data distribution. At all doses (50, 100 and 150mg/kgb.w.), after acute treatment with isatin, alterations in DNA migration (comet assay) were not observed and mutagenic effects were not seen (micronucleus test on peripheral blood cells). After repeated doses, only the highest dose of isatin (150mg/kgb.w.) induced alterations in the DNA that gave rise to micronuclei in the bone marrow and peripheral blood cells of the mice. Our results show that the mutagenic and genotoxic effects of isatin depend on dose and on period of exposure.     

Species differences in the genotoxicity of cyclophosphamide and styrene in three in vivo assays.

Species differences in dispositional factors such as distribution, metabolism and excretion may often account for species differences in the toxic responses to foreign chemicals. In this study we compared the genotoxic responses of cyclophosphamide (CP) and styrene (ST) between Porton rats and LACA Swiss mice in three in vivo assays (bone marrow micronucleus (MN), sperm morphology (SM) and sister-chromatid exchange (SCE) assays). The sensitivities of the three assays were compared by the doses of the compounds required to elicit a significant genotoxic response. The baseline levels for the MN, SCE and SM assays were 1.1-1.4 and 1.2-1.3 MNPCEs/1000 PCEs, 0.23-0.24 and 0.20-0.21 SCEs/chromosome, 3.5-5.7% and 1.6-1.9% abnormal sperm in mice and rats, respectively. CP was a potent genotoxin in the MN and SCE assays but weakly genotoxic in the SM assay. At comparable doses, the rat was approximately 3-, 2.5- and 1.8-fold more sensitive to CP than mice in the MN, SM and SCE assays, respectively. ST produced weak genotoxic responses in all assays in mice and only in the SM and SCE assays in rats. The mice were more sensitive to ST in the MN and SM assays, while it was difficult to compare the species in the SCE assay. For both compounds the sensitivity of the three assays, in decreasing order, were SCE greater than MN much greater than SM. For CP the relative responses in the Porton rats and LACA Swiss mice were qualitatively similar to previous reports. Although the use of different strains may explain differences between the studies in the magnitude of the responses observed. The results for ST in the rat shows that the choice of genotoxic endpoint can determine whether a response is detectable. Moreover, the discrepancies between the results for ST in this study and others, suggest that as well as using a battery of in vivo tests, it may be prudent to select more that one strain or species to fully assess a compound's ability to produce DNA damage.