Determination of the Environmental Pollution Potential of Some Herbicides by the Assessment of Cytotoxic and Genotoxic Effects on Allium cepa.

The present study aims to evaluate the potential for the pollution of the environment by two herbicides (quizalofop-p-ethyl and cycloxydim), using the Allium test. The species in question is Allium cepa (onion, 2n = 16), one of the most common plant indicators of environmental pollution. The working method consisted of obtaining the meristematic roots of Allium cepa and their treatment with herbicides at three different concentrations (0.5%, 1%, and 1.5%) for each herbicide for 24 h, for comparison with an untreated control. The results obtained from the cytological study indicated a strong cytotoxic and genotoxic effect for both herbicides, but especially for quizalofop-p-ethyl, where the mitotic index decreased from 30.2% (control) to 9.6% for the variant treated with 1.5% herbicide. In this case, a strong mitodepressive effect was shown by a highly significant percentage (35.4%) of chromosomal aberrations and nuclear alterations: stickiness, fragments, C-mitosis, lobulated nucleus, micronuclei, and nuclear erosion. The mitodepressive effect as well as the percentage of chromosomal aberrations increased with a higher herbicide concentration. The obtained results suggest the strong potential for pollution of the two herbicides, particularly at concentrations higher than 0.5%; therefore, we recommend caution in their use to avoid undesirable effects on the environment.

Qualitative and quantitative approaches in the dose-response assessment of genotoxic carcinogens.

Qualitative and quantitative approaches are important issues in field of carcinogenic risk assessment of the genotoxic carcinogens. Herein, we provide quantitative data on low-dose hepatocarcinogenicity studies for three genotoxic hepatocarcinogens: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and N-nitrosodiethylamine (DEN). Hepatocarcinogenicity was examined by quantitative analysis of glutathione S-transferase placental form (GST-P) positive foci, which are the preneoplastic lesions in rat hepatocarcinogenesis and the endpoint carcinogenic marker in the rat liver medium-term carcinogenicity bioassay. We also examined DNA damage and gene mutations which occurred through the initiation stage of carcinogenesis. For the establishment of points of departure (PoD) from which the cancer-related risk can be estimated, we analyzed the above events by quantitative no-observed-effect level and benchmark dose approaches. MeIQx at low doses induced formation of DNA-MeIQx adducts; somewhat higher doses caused elevation of 8-hydroxy-2'-deoxyquanosine levels; at still higher doses gene mutations occurred; and the highest dose induced formation of GST-P positive foci. These data indicate that early genotoxic events in the pathway to carcinogenesis showed the expected trend of lower PoDs for earlier events in the carcinogenic process. Similarly, only the highest dose of IQ caused an increase in the number of GST-P positive foci in the liver, while IQ-DNA adduct formation was observed with low doses. Moreover, treatment with DEN at low doses had no effect on development of GST-P positive foci in the liver. These data on PoDs for the markers contribute to understand whether genotoxic carcinogens have a threshold for their carcinogenicity. The most appropriate approach to use in low dose-response assessment must be approved on the basis of scientific judgment.

Evaluation of the safety of primary metabolites of cyadox: Acute and sub-chronic toxicology studies and genotoxicity assessment.

Cyadox (CYA) is a synthetic antimicrobial agent, belonging to quinoxaline (QdNO) family. Cy1 (bidesoxy cyadox), Cy2 (N4-desoxycyadox) and Cy10 (N1-desoxycyadox) are the primary metabolites of CYA. In our present study, an acute toxicity test, a sub-chronic toxicity test, and a battery of three genotoxicity tests were carried out according to standard protocols. The LD50 of the metabolites were above 5000 mg/kg b.w. The maximum tolerated dose (MTD) of Cy1 and Cy-M (mixture of Cy2 and Cy10) in rats, and the MTD of Cy1, Cy2 and Cy10 in mice were above 6000 mg/kg b.w./day. In subchronic study, rats were separately administered Cy1 and Cy-M at the dose levels of 0, 50, 150 and 2500 mg/kg diet for 90 days, with CYA (2500 mg/kg) as a control. Significant decreases in body weight and changes in clinical serum biochemistry were observed in the high-dose group of Cy1 and Cy-M, as well as CYA. Significant changes in relative weights of organs at 150 and 2500 mg/kg diet of Cy1 and CYA were noted. Additionally, the high-dose groups of Cy1, Cy-M and CYA showed pathological changes near the hepatic portal area. There was no evidence for genotoxic activity of any of the three metabolites in the bacterial reverse mutation test, mouse bone marrow micronucleus assay or an in vitro assay for clastogenicity. Based on the subchronic study, the target organ of the primary metabolites was the liver, and the no-observed-adverse-effect level for Cy1 and Cy-M was 150 mg/kg diet.

Assessment of thirteen-week subchronic oral toxicity of cyadox in Beagle dogs.

Cyadox (2-formylquinoxaline-N(1),N(4)-dioxide cyanocetylhydrazone) is a new antimicrobial agent and growth-promoter to be used in food-producing animals. Although its toxicity has been clearly documented in rodents, no study is available in non-rodent animals. Therefore, we studied the subchronic effects of cyadox in Beagle dogs to provide additional information with which to establish safety criteria for human exposure. For this purpose, 36 Beagle dogs, 18 males and 18 females, were divided into four groups and fed diets containing 0, 100, 450 and 2500 mg/kg of cyadox, respectively, for 13 weeks. It was found that there were no significant changes among the examined parameters, except for an increase in the level of serum potassium (K(+)) in 2500 mg/kg cyadox group in males at week 13 of the study. However, the K(+) level returned to normal during the recovery period. In conclusion, cyadox showed slight effects in Beagle dogs in the subchronic oral toxicity study. The no-observed-adverse-effect level of cyadox was considered to be 450 mg/kg diet, which equates to approximately 15.3-15.4 mg/kg b.w./day. The study provided subchronic effects of cyadox in Beagle dogs, suggesting that cyadox might present mild toxicity in non-rodents.

Comparative pathophysiology, toxicology, and human cancer risk assessment of pharmaceutical-induced hibernoma.

In humans, hibernoma is a very rare, benign neoplasm of brown adipose tissue (BAT) that typically occurs at subcutaneous locations and is successfully treated by surgical excision. No single cause has been accepted to explain these very rare human tumors. In contrast, spontaneous hibernoma in rats is rare, often malignant, usually occurs in the thoracic or abdominal cavity, and metastases are common. In recent years, there has been an increased incidence of spontaneous hibernomas in rat carcinogenicity studies, but overall the occurrence remains relatively low and highly variable across studies. There have only been four reported examples of pharmaceutical-induced hibernoma in rat carcinogenicity studies. These include phentolamine, an alpha-adrenergic antagonist; varenicline, a nicotine partial agonist; tofacitinib, a Janus kinase (JAK) inhibitor; and hydromorphone, an opiod analgesic. Potential non-genotoxic mechanisms that may contribute to the pathogenesis of BAT activation/proliferation and/or subsequent hibernoma development in rats include: (1) physiological stimuli, (2) sympathetic stimulation, (3) peroxisome proliferator-activated receptor (PPAR) agonism, and/or (4) interference or inhibition of JAK/Signal Transducer and Activator of Transcription (JAK/STAT) signaling. The evaluation of an apparent increase of hibernoma in rats from 2-year carcinogenicity studies of novel pharmaceutical therapeutics and its relevance to human safety risk assessment is complex. One should consider: the genotoxicity of the test article, dose/exposure and safety margins, and pathophysiologic and morphologic differences and similarities of hibernoma between rats and humans. Hibernomas observed to date in carcinogenicity studies of pharmaceutical agents do not appear to be relevant for human risk at therapeutic dosages.

In vivo genotoxicity and cytotoxicity assessment of a novel quinoxalinone with trichomonacide activity.

The compound VAM2-6 (1-methyl-7-nitro-4-(5-(piperidin-1-yl)pentyl)-3,4-dihydroquinoxalin-2(1H)-one) has previously been shown to have an in vitro efficacy of 100% at a concentration of 100 µg ml(-1) against Trichomonas vaginalis, a protozoon parasite that causes the sexually transmitted disease trichomoniasis. Because VAM2-6 is a quinoxaline derivative and given the lack of studies on the genotoxic activity of this compound, the present study was undertaken to evaluate its ability to induce DNA damage in the peripheral blood of mice using single-cell gel electrophoresis (SCGE or comet assay) and the micronucleus (MN) assay. Cell viability was assessed using a fluorochrome-mediated viability test. The compound was tested on CD1 mice at 60, 40 and 10 mg kg(-1) body weight administrated intraperitoneal (i.p.) in a single dose. Peripheral blood samples were collected 24 and 48 h after treatment. N-Ethyl-N-nitrosourea (ENU) was used as a positive control for the comet and micronucleus assays. The results showed that i.p. VAM2-6 induced single-strand DNA breaks and increased the average number of micronuclei in the treated mice in a dose-dependent manner at 60, 40 and 10 mg kg(-1). Cell viability decreased at 24 h but recovered at 48 h for all three evaluated doses. Therefore, the chemical structure of VAM2-6 should be modified to reduce its genotoxic potential.

Differential enantioselectivity of quizalofop ethyl and its acidic metabolite: direct enantiomeric separation and assessment of multiple toxicological endpoints.

Transformation products usually differ in environmental and toxicological properties compared to the parent contaminants, thus causing potential and unknown environmental risks. To elucidate differential chiral recognition of the aryloxypropanoate herbicide quizalofop ethyl (QE) and its primary product (quizalofop acid, QA), their enantiomeric separation and toxicological impacts to two freshwater algae were investigated. Addition of trace water (0.02-0.08%, v/v) to the mobile phase selectively affected retention of analyte and induced simultaneous enantio-separation for the two compounds with intrinsical water-specific resolution mechanisms, although they both possessed a chiral center in the 2-position of propionates. In algal suspensions, QE was rapidly degraded to produce the acid metabolite (QA), and the product further declined, whereas a reduction of QA as starting compound did not occur. Uptake and/or transformation of QE and QA were found a lack of enantioselectivity and isomer inversion, while cellular membrane permeability, membrane potential and algal growth showed enantioselectivity to different extents. These results suggested the presence of receptor chirality that was involved in the toxicological processes but invalid for uptake and transformation. Therefore, quizalofop acid, identified as environmentally relevant contaminant associated with application of the herbicide, participated in the toxicological processes of the parent compound, and exhibited distinct toxicological and chromatographic retention properties.

Toxicity assessment of herbicides quizalafop-p-ethyl and clodinafop towards Rhizobium pea symbiosis.

In modern conventional agriculture, herbicides are frequently used to prevent yield losses due to weeds. Herbicides also affect negatively the productivity of legumes. With these considerations, we evaluated the effects of soil applications of different concentrations of quizalafop-p-ethyl and clodinafop on the performance of Rhizobium inoculated pea, grown in clay pots. In this study, the concentration of herbicides higher than the recommended rates of quizalafop-p-ethyl and clodinafop adversely affected the dry matter accumulation, symbiotic properties, grain yield and nutrient status of pea plants. Toxicity of quizalafop-p-ethyl and clodinafop to pea plants increased progressively with increase in rates of herbicides. Of the two herbicides, quizalafop-p-ethyl was more toxic than clodinafop. In contrast, when herbicide tolerant Rhizobium strain MRP1 was also used with herbicide, it increased the measured parameters at all concentrations. A maximum increase of 11%, 17%, 46%, 33%, 21% and 7% in the root N, shoot N, root P, shoot P, seed yield and seed protein, respectively, was observed when MRP1 was used with 120 microg quizalafop-p-ethyl kg(-1) soil while with 1,200 microg clodinafop kg(-1) soil it increased the root N, shoot N, root P, shoot P, seed yield and seed protein by 20%, 9%, 56%, 56%, 29% and 7%, respectively, compared with the un-inoculated but herbicide treated control. This study suggested that the toxic effects of herbicides on pea plants could be attenuated by applying growth promoting herbicide tolerant strain of Rhizobium under herbicide stressed soil environment.

Carcinogenic risk of heterocyclic amines in combination - assessment with a liver initiation model.

Carcinogenic potential of heterocyclic amines (HCAs) was investigated using an in vivo 5-week initiation assay with quantitative evaluation of glutathione S-transferase placental form (GST-P) positive foci in rat liver. Numbers of GST-P positive foci were significantly increased with individual administration of six different HCAs, indicating utility of the assay. It was therefore applied to investigate risk with multiple HCAs in combination. Unexpectedly, concomitant treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) did not result in any additive carcinogenicity. In the rats taking MeIQx prior to PhIP the value was almost equal to the sum total of individual data, indicating additive initiation activities. In contrast, simultaneous or prior administration of PhIP rather exerted inhibitory effects on the carcinogenic potential of MeIQx. Moreover, microarray and quantitative RT-PCR assessment revealed that PhIP induced cytochrome P450 1A1, responsible for both activation and detoxification of HCAs, more strongly than MeIQx. It is noteworthy that complex exposure to multiple HCAs is not necessarily associated with increased risk of carcinogenesis because they are simultaneously and continuously ingested under normal circumstances.

Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound.

AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high receptor affinity, good in vivo potency, and oral activity. AMP397 has a structural alert (aromatic nitro group) and was mutagenic in Salmonella typhimurium strains TA97a, TA98 and TA100 without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. The amino derivative of AMP397 was negative in wild-type strains TA98 and TA100. AMP397 was negative in a mouse lymphoma tk assay, which included a 24h treatment without S9. A weak micronucleus induction in vitro was found at the highest concentrations tested in V79 cells with S9. AMP397 was negative in the following in vivo studies, which included the maximum tolerated doses of 320mg/kg in mice and 2000mg/kg in rats: MutaMouse assay in colon and liver (5x320mg/kg) at three sampling times (3, 7 and 31 days after the last administration); DNA binding study in the liver of mice and rats after a single treatment with [14C]-AMP397; comet assay (1x2000mg/kg) in jejunum and liver of rats, sampling times 3 and 24h after administration; micronucleus test (2x320mg/kg) in the bone marrow of mice, sampling 24h after the second administration. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. In particular, no genotoxic metabolite is formed in mammalian cells, and, if formed by intestinal bacteria, is unable to exert any genotoxic activity in the adjacent intestinal tissue. These data were considered to provide sufficient safety to initiate clinical development of the compound.

Analysis and quantification of DNA adducts of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in liver of rats by liquid chromatography/electrospray tandem mass spectrometry.

Liquid chromatography with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to measure DNA adducts of the carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) with a microbore C-18 reversed-phase column. Quantification of the isomeric adducts N-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-C8-MeIQx) and 5-(deoxyguanosin-N(2)-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-N(2)-MeIQx) was achieved using synthetic, isotopically labeled internal standards. The reaction of the N-acetoxy ester of 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline (HONH-MeIQx) with calf thymus DNA (ct DNA) resulted in formation of these adducts in a ratio of 5:1 (dG-C8-MeIQx:dG-N(2)-MeIQx). The detection limit by LC/ESI-MS/MS in the selected reaction monitoring (SRM) mode ([MH(+) --> MH - 116](+)) (loss of deoxyribose) approached 500 fg (1 fmol) of adduct standard, and 1 adduct per 10(8) DNA bases using 100 microg of DNA following solid-phase extraction. The SRM analysis of rat liver DNA 24 h after an oral dose of MeIQx (10 and 0.5 mg/kg) revealed the presence of isomeric dG-MeIQx adducts at levels of 3.07 +/- 0.84 and 0.45 +/- 0.27 adducts per 10(7) bases, respectively. LC/ESI-MS/MS product ion spectra were acquired on both adducts from the elevated dose of MeIQx for unambiguous adduct identification. The contribution of dG-N(2)-MeIQx to the total adducts in vivo was significantly more important than that observed in vitro. dG-C8-MeIQx was the principal adduct formed at the 10 mg/kg dose, (dG-C8-MeIQx:dG-N(2)-MeIQx (3:2)); however, dG-N(2)-MeIQx was the major lesion detected at the 0.5 mg/kg dose (dG-C8-MeIQx:dG-N(2)-MeIQx 1:10). The striking differences between the relative amounts of dG-C8-MeIQx and dG-N(2)-MeIQx formed in vivo as a function of dose suggest that reactive esters of HONH-MeIQx other than N-acetoxy-MeIQx may be formed in vivo and react preferentially with the N(2) atom of guanine, or that dG-C8-MeIQx is removed at a significantly more rapid rate than dG-N(2)-MeIQx. The dG-N(2)-MeIQx adduct, previously thought to be a minor adduct, is likely to be an important contributor to the genotoxic damage of MeIQx.

[The use of the fluorimetric method for studying DNA integrity exemplified by an assessment of dioxidine genotoxicity]. / Primenenie fliuorimetricheskogo metoda issledovaniia tselostnosti DNK na primere otsenki genotoksichnosti dioksidina.

The genotoxic effect of dioxidine (300, 100, 30, and 10 mg/kg, i.p.) on the liver cells of outbred male rats was studied by fluorometric analysis of DNA unwinding (FADU) using the cell DNA samples taken from animals sacrificed 1, 6, 18, and 24 h after drug introduction. At a dose of 10 mg/kg, dioxidine produced no statistically significant increase in the amount of DNA damage relative to control. Increase in the dioxidine dose to 30 mg/kg resulted in a significant DNA damage 1 h after drug administration, while a dose of 100 or 300 mg/kg reliably caused damage at all times of exposure.

Low-dose carcinogenicity of a heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline: relevance to risk assessment.

Male, 21-day-old, F344 rats were administered 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in the diet at various low doses and a high dose, 100 ppm for 16 weeks. Quantitative values for glutathione-S-transferase placental form (GST-P)-positive foci in their livers were similar among the 0, 0.001, 0.01, 0.1 and 1 ppm MeIQx group while 10 ppm MeIQx administration slightly and 100 ppm MeIQx significantly increased their numbers. These results indicate that MeIQx has a no-observed effect level for induction of preneoplastic lesions in rats. Transplacental and trans-breast milk exposure to low doses of MeIQx also did not exert carcinogenic potential in F344 rats and 20% of calorie restriction clearly inhibited development of GST-P-positive foci. The results are of direct significance to human risk assessment.

Carcinogenic risk assessment of MeIQx and PhIP in a newborn mouse two-stage tumorigenesis assay.

A newborn mouse two-stage tumorigenesis assay was evaluated as a possible alternative to chronic rodent carcinogenicity bioassays by investigating the carcinogenicity of two major heterocyclic amines, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). One week after birth, Crj:CD-1 mice of both sexes were subcutaneously administered N-nitrosobis(2-oxopropyl)amine (BOP) at a dose of 50 mg/kg as an initiation treatment and starting 2 weeks thereafter they were fed diets supplemented with MeIQx at concentrations of 300, 30, 3 or 0 ppm or PhIP at 200, 50, 10 or 0 ppm for 23 weeks. Animals in all groups predominantly developed tumors of the lung and liver. Pulmonary adenomas and adenocarcinomas were observed in all groups with high incidences, without any significant differences between the groups. MeIQx and PhIP did not influence the multiplicity except in the group given 10 ppm PhIP where it significantly increased the number of pulmonary adenomas (P < 0.05). Similarly, hepatocellular adenomas and carcinomas were found in all groups with high incidences, and again MeIQx and PhIP did not increase their incidences or multiplicities. Although adenomas and carcinomas in the nasal cavity, and acinar cell foci in the pancreas were infrequently found, none of these incidences were increased with the MeIQx or PhIP treatments. These results thus suggest that the tumor-promoting effects of MeIQx or PhIP may be rather weak, if present, as far as this newborn two-stage model is concerned, and the possibility that the dose of BOP was too high to detect the tumor-promoting effects of MeIQx or PhIP by masking them cannot be completely ruled out.