Aberrant cell cycle regulation in rat liver cells induced by post-initiation treatment with hepatocarcinogens/hepatocarcinogenic tumor promoters.

The present study aimed to determine the onset time of hepatocarcinogen/hepatocarcinogenic tumor promoter-specific cell proliferation, apoptosis and aberrant cell cycle regulation after post-initiation treatment. Six-week-old rats were treated with the genotoxic hepatocarcinogen, carbadox (CRB), the marginally hepatocarcinogenic leucomalachite green (LMG), the tumor promoter, ß-naphthoflavone (BNF) or the non-carcinogenic hepatotoxicant, acetaminophen, for 2, 4 or 6 weeks during the post-initiation phase using a medium-term liver bioassay. Cell proliferation activity, expression of G2 to M phase- and spindle checkpoint-related molecules, and apoptosis were immunohistochemically analyzed at week 2 and 4, and tumor promotion activity was assessed at week 6. At week 2, hepatocarcinogen/tumor promoter-specific aberrant cell cycle regulation was not observed. At week 4, BNF and LMG increased cell proliferation together with hepatotoxicity, while CRB did not. Additionally, BNF and CRB reduced the number of cells expressing phosphorylated-histone H3 in both ubiquitin D (UBD)(+) cells and Ki-67(+) proliferating cells, suggesting development of spindle checkpoint dysfunction, regardless of cell proliferation activity. At week 6, examined hepatocarcinogens/tumor promoters increased preneoplastic hepatic foci expressing glutathione S-transferase placental form. These results suggest that some hepatocarcinogens/tumor promoters increase their toxicity after post-initiation treatment, causing regenerative cell proliferation. In contrast, some genotoxic hepatocarcinogens may disrupt the spindle checkpoint without facilitating cell proliferation at the early stage of tumor promotion. This suggests that facilitation of cell proliferation and disruption of spindle checkpoint function are induced by different mechanisms during hepatocarcinogenesis. Four weeks of post-initiation treatment may be sufficient to induce hepatocarcinogen/tumor promoter-specific cellular responses.

Disruption of spindle checkpoint function ahead of facilitation of cell proliferation by repeated administration of hepatocarcinogens in rats.

We aimed to clarify the hepatocarcinogen-specific disruption of cell cycle checkpoint functions and its time course after repeated administration of hepatocarcinogens. Thus, rats were repeatedly administered with hepatocarcinogens (methapyrilene, carbadox and thioacetamide), a marginal hepatocarcinogen (leucomalachite green), hepatocarcinogenic promoters (oxfendazole and ß-naphthoflavone) or non-carcinogenic hepatotoxicants (promethazine and acetaminophen) for 7, 28 or 90 days, and the temporal changes in cell proliferation, expression of G1/S and spindle checkpoint-related molecules, and apoptosis were examined using immunohistochemistry and/or real-time RT-PCR analysis. Hepatocarcinogens facilitating cell proliferation at day 28 of administration also facilitated cell proliferation and apoptosis at day 90. Hepatocarcinogen- or hepatocarcinogenic promoter-specific cellular responses were not detected by immunohistochemical single molecule analysis even after 90 days. Expression of Cdkn1a, Mad2l1, Chek1 and Rbl2 mRNA also lacked specificity to hepatocarcinogens or hepatocarcinogenic promoters. In contrast, all hepatocarcinogens and the marginally hepatocarcinogenic leucomalachite green induced Mdm2 upregulation or increase in the number of phosphorylated MDM2(+) cells from day 28, irrespective of the lack of cell proliferation facilitation by some compounds. However, different Tp53 expression levels suggest different mechanisms of induction or activation of MDM2 among hepatocarcinogens. On the other hand, hepatocarcinogenic methapyrilene and carbadox downregulated the number of both ubiquitin D(+) cells and proliferating cells remaining in M phase at day 28 and/or day 90, irrespective of the lack of cell proliferation facilitation in the latter. These results suggest that hepatocarcinogens disrupt spindle checkpoint function after 28 or 90 days of administration, which may be induced ahead of cell proliferation facilitation.

Genotoxicity evaluation of Mequindox in different short-term tests.

Quinoxaline-1,4-dioxides (QdNOs) are the potent heterocyclic N-oxides with interesting biological properties such as antibacterial, anticandida, antitubercular, anticancer and antiprotozoal activities. Here, we tested and compared the mequindox (MEQ) for mutagenic abilities in a battery of different short term tests according to OECD guidelines. When compared with the controls, a strong mutagenicity of MEQ and carbadox (CBX) was observed with an approximate concentration-effect relationship in Salmonella reverse mutation test, chromosome aberration test, unscheduled DNA synthesis assay and HGPRT gene mutation test, in the absence and presence of S(9)-mix. In in vivo micronucleus test, CBX produced significant increase in the proportion of micronucleus formation than MEQ in mice bone marrow cells. From these results, we can conclude that MEQ had a strong genotoxic potential to mammalian cells in vitro as well as in vivo and its mutagenicity is slightly higher than CBX. Our results, for the 1st time, discuss the genotoxic potential of MEQ. These results not only confirm the earlier findings about CBX but also extend the knowledge and awareness about the genotoxic risk of QdNO derivatives.

Investigation of the genotoxicity of quinocetone, carbadox and olaquindox in vitro using Vero cells.

Quinoxaline-1,4-dioxides derivatives have been widely used as animal growth promoter. This study was conducted to investigate the cytotoxicity and genotoxicity of quinoxaline-1,4-dioxides derivatives, namely carbadox, olaquindox and quinocetone, in Vero cells. The cell viability results from MTT assay demonstrated the severe inhibitory effects by these chemicals in both dose and time dependent manner. Among these chemicals quinocetone exhibited the highest cytotoxicity followed by olaquindox and carbadox. DNA damage analyses using alkalic comet assay revealed pronounced increase of DNA fragmentation in all three compound treated cells. In contrast, DNA damage was significantly decreased after incubation with S9 mix. These findings suggest that the intermediate metabolites of these compounds exerted lower genotoxicity than their parent drugs. We further described chromosomal damage induced by these drugs employing cytokinesis-block micronucleus assay (MN assays). The micronucleus frequency was significantly higher in these drugs treated cells than that of controls and the nuclear division index was also markedly reduced with increasing drug concentration applied. Similar to the observation in comet assay, incorporation of S9 mix in the MN assays was able to markedly alleviate the chromosome damage. In conclusion, our results strengthened previous reports on the cytotoxicity and genotoxicity of carbadox, olaquindox and quinocetone.

Characterization of carbadox-induced mutagenesis using a shuttle vector pSP189 in mammalian cells.

Carbadox, a quinoxaline 1,4-dioxide derivative, is a known mutagen with its functional mechanism yet to be well defined. In the present study we used a shuttle vector assay in vitro to uncover the functional details of carbadox-induced mutagenesis in mammalian cells. The plasmid DNA of a shuttle vector pSP189 was treated with different doses of carbadox at 37 degrees C for 1 or 2h with or without the presence of S9. The target gene SupF in the plasmid was sequenced after replication in Vero cells followed by amplification in Escherichia coli MBM7070 to evaluate mutation frequency. DNA sequencing analysis of recovered carbadox-induced mutations revealed 76.3% single base substitution, 7.9% single base insertion, 10.5% single base deletion and 5.3% large fragments deletion. All single base substitutions occurred at G:C base pairs, among which transversion and transition occurred at a 2:1 ratio. The mutations did not occur randomly in the supF gene, but had sequence specificity and hotspots instead: most substitutions were detected at the nucleotide N in a 5'-NNTTNN-3' sequence; 75% of base insertions were seen in the 5'-TCC-3' sequence; whereas all large fragments deletions occurred in the 5'-ANGGCCNAAA-3' sequence. Nucleotide 129, 141 and 155 in the supF gene of plasmid pSP189 were identified as the hotspots for carbadox-induced mutations that accounted for 65% of all single base substitutions. We conclude that carbadox and its metabolites induce sequence-specific DNA mutations at high frequencies, therefore its safe usage in animal husbandry should be seriously considered.

Teratogenic assessment of carbadox in rats.

Carbadox was administered by gavage once daily to pregnant rats at doses of 0 (control), 10, 25, 50 or 100 mg/kg on days 8 through 15 of pregnancy. The dams were killed on day 21 and the number of implantation sites, resorptions and live fetuses were counted. A significant dose-related decrease in maternal body weight gains during treatment (days 8 through 15 of pregnancy) occurred at doses of 10 mg/kg and above. There was a dose-related decrease in fetal body weights which was statistically significant at 25 mg/kg and above. This compound showed not only embryolethal but teratogenic effect. Resorption rates were 81.8% at 100 mg/kg, occurring complete resorptions in five dams, compared with 3.4% resorption rate in the control. In fetal examinations, a significant increase in the incidence of external, skeletal and internal malformations occurred at 100 mg/kg, where the surviving fetuses born to dams with 40-93% resorptions had any malformations, short tail; kinky tail; brachygnathia or ectrodactyly.

[Toxicological considerations in the evaluation of veterinary drugs]. / Toxicologische overwegingen bij de beoordeling van diergeneesmiddelen.

Interactions between veterinary pharmacotherapy, toxicology of residues, prevention of residues of veterinary drugs and the evaluation of veterinary drug files are discussed on the basis of a number of examples. Sulphadimidine is used to treat atrophic rhinitis in medicated feeds which do not benefit the animal but are the cause of persistent sulphonamide residues in feed mills and husbandry. Carbadox is a potentially effective prophylactic feed additive for the prevention of swine dysentery, but is mostly used in high dosages which are almost toxic for the animals, and used during unnecessary prolonged periods. It is also prescribed as a therapeutic agent in which case a symptom of poisoning, dry faeces, is mistaken for a sign of recovery. Carbadox and/or its metabolites are carcinogenic and its use should be restricted to a bare minimum. Furazolidone is an example of an effective veterinary drug, the use of which should be limited by the fact that detoxification mechanisms of the animals, may result in the appearance of reactive metabolites which are available in the gastro-intestinal tract of the consumer. The central issue in a 'minimal residue' policy regarding the use of veterinary drugs should be the selection of effective drugs. Such a selection could result in a significant reduction of the incidence of veterinary drug residues. Second to this issue is the question of the extent to which residue toxicology should modulate the use of veterinary drugs.

Photochemical reactions of quindoxin, olaquindox, carbadox and cyadox with protein, indicating photoallergic properties.

Quindoxin (quinoxaline-1,4-dioxide), a former 'growth promoter' used in animal husbandry, has been taken from the market because of its photoallergic properties. Nowadays its derivatives olaquindox, carbadox and cyadox are frequently applied for the same purpose. Recent reports show that olaquindox too, can induce photoallergic skin reactions in stockmen. From the present investigation it appeared that all compounds mentioned, form a reactive oxaziridine upon exposure to light, just like many other imino-N-oxides. Photoreactivity with protein, which is considered as an important condition for a compound to be a potential photoallergen, was also studied. Quindoxin and olaquindox proved to meet this condition, as was expected. But carbadox and cyadox also react and were shown to be even more reactive towards human serum albumin.

Comparative study of the effect of the effect of carbadox, olaquindox and cyadox on aldosterone, sodium and potassium plasma levels in weaned pigs.

To study the effects of olaquindox and cyadox on aldosterone, sodium and potassium in the blood in comparison with the effects of carbadox, weaned pigs were fed these compounds in different doses. Pigs treated with 100 and 200 ppm carbadox showed a significant decline of aldosterone after five and three weeks, respectively, compared with control values. In the 200 ppm group treatment was interrupted at week 4. With olaquindox a continuous, significant decline was found from 50 ppm and above after five weeks, and from 25 ppm and above (but excluding the 100 ppm group), after six weeks. In the cyadox groups a significant decline was measured after six weeks in the 50, 200 and 400 ppm groups. Only the 200 ppm group had an earlier response at three and five weeks. A decrease of sodium to hyponatraemic levels in the carbadox groups was seen after three weeks in the 200, and after five weeks in the 100 ppm group. In the olaquindox groups only the 200 ppm dosage showed a consistent decrease to hyponatraemic levels from four weeks treatment. In the cyadox groups the 200 ppm dosage reached a hyponatraemic level after six weeks. An increase of potassium to hyperkalaemic levels occurred at 100 and 200 ppm carbadox dosage after four and three weeks, respectively, and at 200 ppm olaquindox dosage after four weeks. No hyperkalaemic levels were seen in the cyadox groups. It is concluded that the toxic effect of olaquindox, despite minor differences, is comparable with that of carbadox but that cyadox is less toxic.

Clinical signs and performance of pigs during the administration of different levels of carbadox and after withdrawal.

An experiment was designed to study the clinical effects of carbadox in pigs. Five different carbadox levels were tested namely 25, 50, 100, 150, and 200 ppm. They were compared with a control group fed on a diet without medication. After 10 weeks all pigs received a diet without carbadox till the end of the experiment, 21 weeks after the start. After two weeks of carbadox treatment the first clinical signs were observed in the 200 ppm group. The most obvious effects seen were production of dry faeces and drinking of urine from the floor or from pen-mates. Other signs were a decreased abdominal volume, a pale skin with long withered hair, perverted eating and a restless behaviour. The haematocrit values in pigs receiving 100 ppm and upwards were increased. There was a negative correlation between the dose of carbadox and the time after which the response occurred. Weight gain was significantly lowered and feed conversion essentially poorer in the 200 ppm, 150 ppm and 100 ppm groups during the treatment as compared to the controls. No growth promoting effect was seen in the 25 and 50 ppm groups. After withdrawal of carbadox, clinical signs persisted in the 150 and 200 ppm groups. The 100 ppm group produced normal faeces 5 weeks after withdrawal, whereas drinking of urine persisted. From this study it appears that only an oral dosage of 25 ppm or lower can be given to pigs without risks of toxic effects. The widely claimed growth promoting effect of carbadox could not be confirmed in this study. This might be due to the small number of animals per group.

Hypoaldosteronism in piglets induced by carbadox.

An exploratory study was made of the mechanisms underlying the toxic action of carbadox in young pigs: dehydration, loss of appetite and at autopsy seemingly specific and selective structural alterations of the glomerular zone of the adrenal cortex. Administration of carbadox in the feed, in dosages of 150 ppm (approximately 6 mg X kg-1 b. wt X day-1) caused a rapid decline in the plasma aldosterone levels (to 10% of control) followed by significant changes in the sodium and potassium levels in blood. Characteristic for the toxic action of carbadox are the rapid and seemingly selective and specific alterations in the aldosterone-releasing zona glomerulosa of the adrenals. Our results indicate that with carbadox a functional and possibly reversible extirpation of the adrenal zona glomerulosa can be achieved in pigs.

[Pharmacology and toxicology of furazolidone and carbadox; a literature study]. / Farmacologie en toxicologie van furazolidon en carbadox; een literatuuronderzoek.

A literature search on furazolidone in pigs was made in order to be able to answer a number of questions raised by a court regarding possible furazolidone intoxication. The following review of the literature is the result of this study. Attention is also paid to the residual toxicology of this drug. As one of the court's questions concerned carbadox alone and combined with furazolidone, a brief review of the pharmacology of this drug is included.

Cytogenetic effects of quinoxaline-1,4-dioxide-type growth-promoting agents. II. Metaphase analysis in mice.

The clastogenicity of the growth-promoting agents, carbadox, olaquindox and cyadox, was examined by the metaphase analysis of chromosome aberrations in bone marrow of mice. The agents were administered by a stomach tube to male ICR mice 24 h before they were killed. Single-dose levels were 50, 100 and 200 mg/kg for carbadox; 200, 400, 600 and 800 mg/kg for olaquindox; and 400, 800 and 1200 mg/kg for cyadox. Carbadox was the most active; it induced chromosome aberrations even at a dose of 100 mg/kg. Olaquindox had about the same activity at a dose of 800 mg/kg. No chromosome-damaging effect of cyadox was observed even at a dose of 1200 mg/kg.

Negative results of carcinogenicity bioassay of methyl carbazate in rats: significance for the toxicological evaluation of carbadox.

Methyl carbazate, a metabolite of carbadox in the rat, was administered orally to rats for 2 years. The compound was mixed in the diet at concentrations corresponding to dose levels of 0, 2.5, 5 and 10 mg/kg body wt/day. There was no evidence of any treatment-related toxicity or, in particular, of carcinogenicity.

[Toxicity by relay. III. Safety for the human consumer of the use of Carbadox, a feed additive for swine, as estimated by a 7 years relay toxicity on dogs]. / La toxicite de relais III. securite d'emploi pour le consommateur human du carbadox, facteur de croissance pour le porc, estimee par toxicite de relais d'une duree de sept ans chez le chien beagle.

To check the possible toxicity risks of the meat from swine fed with a residue producing feed additive: Carbadox, a new methodology was used. It is the "Relay Toxicity". Swine were fed with a high dose of the additive and sacrificed without any withdrawal. In such conditions meat contains a high level of residues. The frozen meat was given daily--after thawing--at 100 g or 200 g/dog to 12 beagle dogs, 6 females and 6 males, sacrificed when 87.5 months old (85 months on experiment). On these animals, no anomaly was found: on weight gain and health; on fertility and reproductive performances; on hematology and biochemical values of blood and urine; and after careful macroscopic or microscopic examinations of the animals at safrice. We were able to obtain a safety factor above 9000 when Carbadox is used at the maximum level approved 50 ppm, and the withdrawal 4 weeks before slaughtering. To conclude, the absence of anomaly allows us to confirm the safety for the human consumer when Carbadox is used as a feed additive for swine.

Principles of a full 'relay toxicity' experiment and results conducted with carbadox, a feed additive used as growth promoter for growing swine.

Relay toxicity is a new approach which permits an evaluation of the harmlessness of residues found in tissue, for the human consumer. Farm animals, swine for example, received high quantities of the additive over a long period. The swine were sacrificed without any withdrawal. Their meat and liver were added to the feed ration of laboratory animals to study the eventual problems of residue and metabolites. The following experiments were performed using carbadox:--Levels of carbadox up to 200 ppm were added to the swine ration (a maximum of 20 ppm is authorized). The meat and dehydrated liver were given in doses of 20% and 10% respectively, in the rat feed for 2 years or 3 generations.--The fresh meat, frozen then thawed at the time of use, was distributed to dogs daily for 7 1/2 years. No abnormalities were found, either in the growth of the animals or in their descendants. No abnormalities were observed macroscopically or microscopically after sacrifice. Relay toxicity gives high coefficients of security when the additive is used without withdrawal up to the time of sacrifice. This study demonstrated that the use of carbadox in swine doesn't present any disadvantage to the human consumer.