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1.
J Toxicol Sci ; 47(5): 193-199, 2022.
Article in English | MEDLINE | ID: mdl-35527007

ABSTRACT

According to ICH S3A Q&A focusing on microsampling, its application should be avoided in main study animals for test drugs that could exacerbate hematological parameters with frequent blood sampling. However, no study has reported the effects of microsampling on toxicity parameters of drugs known to induce hematological toxicity. Therefore, we assessed the toxicological effects of serial microsampling on rats treated with phenacetin as a model drug. In a common 28-day study, 50 µL of microsampling was performed at 6-time points on days 1 to 2 and 7-time points on days 27 to 28 from the jugular vein of Sprague Dawley rats. The study was performed independently by two organizations. The toxicological influence of microsampling was evaluated on body weight, food consumption, hematology, blood clinical chemistry, urine parameters, organ weights, and tissue pathology. Phenacetin treatments induced significant changes of various hematological parameters (including hemoglobin and reticulocytes), some organ weights (including liver and spleen), and some hematology-related pathological parameters in the liver, spleen and bone marrow. Meanwhile, serial microsampling exhibited minimal influence on the assessed parameters, although 20 parameters showed statistical differences mostly at one organization. The current results support the notion that serial 50 µL microsampling from the jugular vein had minimal impacts on overall toxicological profiles even in rats treated with a drug inducing hematological toxicity, but the potential adverse effect on certain parameters could not be fully excluded. Accordingly, this microsampling technique has possibility to be employed even for non-clinical rat toxicity studies using drugs with potentially hematological toxicity.


Subject(s)
Blood Specimen Collection , Phenacetin , Animals , Blood Specimen Collection/methods , Body Weight , Jugular Veins , Phenacetin/toxicity , Rats , Rats, Sprague-Dawley , Spleen
2.
Ecotoxicol Environ Saf ; 204: 110977, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32739673

ABSTRACT

Indirect oxidation induced by reactive free radicals, such as hydroxyl radical (HO), sulfate radical (SO4-) and carbonate radical (CO3-), plays an important or even crucial role in the degradation of micropollutants. Thus, the coadjutant degradation of phenacetin (PNT) by HO, SO4- and CO3-, as well as the synergistic effect of O2 on HO and HO2 were studied through mechanism, kinetics and toxicity evaluation. The results showed that the degradation of PNT was mainly caused by radical adduct formation (RAF) reaction (69% for Г, the same as below) and H atom transfer (HAT) reaction (31%) of HO. For the two inorganic anionic radicals, SO4- initiated PNT degradation by sequential radical addition-elimination (SRAE; 55%), HAT (28%) and single electron transfer (SET; 17%) reactions, while only by HAT reaction for CO3-. The total initial reaction rate constants of PNT by three radicals were in the order: SO4- > HO > CO3-. The kinetics of PNT degradation simulated by Kintecus program showed that UV/persulfate could degrade target compound more effectively than UV/H2O2 in ultrapure water. In the subsequent reaction of PNT with O2, HO and HO2, the formation of mono/di/tri-hydroxyl substitutions and unsaturated aldehydes/ketones/alcohols were confirmed. The results of toxicity assessment showed that the acute and chronic toxicity of most products to fish increased and to daphnia decreased, and acute toxicity to green algae decreased while chronic toxicity increased.


Subject(s)
Carbonates/toxicity , Hydrogen Peroxide/toxicity , Phenacetin/toxicity , Sulfates/toxicity , Toxicity Tests, Acute , Toxicity Tests, Chronic , Animals , Carbonates/chemistry , Chlorophyta/drug effects , Daphnia/drug effects , Fishes , Hydrogen Peroxide/chemistry , Ions/chemistry , Ions/toxicity , Kinetics , Models, Chemical , Oxygen/chemistry , Phenacetin/chemistry , Sulfates/chemistry , Water/chemistry
3.
Life Sci ; 228: 176-188, 2019 Jul 01.
Article in English | MEDLINE | ID: mdl-31059688

ABSTRACT

AIM: Inflammatory algesia and pyresia are common pathological consequences of physiological defense. Phenacetin introduced as effective analgesic anti-pyretic agent, was proscribed from therapeutic use because of associated systemic toxicity. The aim of the study was to evaluate the potency of 1,2,3-triazole conjugation in reducing toxicity and increasing efficacy of the phenacetin nucleus. MAIN METHODS: The amide bond implicated as the cause of phenacetin toxicity was bioisosterically replaced with 1,2,3-triazoles to yield a series of PhTCs(PhTC1, PhTC2 and PhTC3). The toxicology of the synthesized conjugates in reference to phenacetin was evaluated in accordance with OECD test guidelines 420, 425 and 407. For the purpose of evaluating anti-inflammatory potency carrageenan induced paw edema and croton oil induced ear edema models were evaluated. Anti-nociceptive efficacy was assessed using Eddy's hot plate and acetic acid induced writhing experimental models. For anti-pyretic efficacy, the conjugates were submitted to Brewer's yeast antipyretic assay. KEY FINDINGS: Toxicological examination of PhTCs in comparison to phenacetin revealed that, phenacetin treatment caused considerable nephrotoxicity and hepatotoxicity in experimental models PhTCs were devoid of such toxic manifestations. Results of pharmacological assays showed that the entire series of PhTCs possessed better anti-inflammatory, anti-nociceptive and anti-pyretic potential than phenacetin. Furthermore it was revealed that the pharmacological profile of PhTC1 with triazole substitution at para position of the phenol ring exhibited potency even better than that exhibited by the reference standards. CONCLUSION: Bioisosteric replacement of amide bond by 1,2,3-triazole in the phenacetin moiety yields conjugates with superior efficacy and diminished toxicity, thus opening neo avenues in treatment of inflammatory syndromes.


Subject(s)
Analgesics/chemistry , Analgesics/pharmacology , Phenacetin/analogs & derivatives , Phenacetin/pharmacology , Triazoles/chemistry , Triazoles/pharmacology , Analgesics/toxicity , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/toxicity , Female , Kidney/drug effects , Kidney/pathology , Liver/drug effects , Liver/pathology , Male , Phenacetin/toxicity , Rats, Wistar , Triazoles/toxicity
4.
Toxicology ; 324: 10-7, 2014 Oct 03.
Article in English | MEDLINE | ID: mdl-25047350

ABSTRACT

Transgenic rat mutation assays can be used to assess genotoxic properties of chemicals in target organs for carcinogenicity. Mutations in transgenes are genetically neutral and accumulate during a treatment period; thus, assays are suitable for assessing the genotoxic risk of chemicals using a repeated-dose treatment paradigm. However, only a limited number of such studies have been conducted. To examine the utility of transgenic rat assays in repeated-dose studies, we fed male and female Sprague-Dawley gpt delta rats with a 0.5% phenacetin-containing diet for 26 and 52 weeks. A long-term feeding of phenacetin is known to induce renal cancer in rats. Phenacetin administration for 52 weeks in males significantly increased gpt (point mutations) mutant frequency (MF) in the kidney, the target organ of carcinogenesis. In the liver, the nontarget organ of carcinogenesis, gpt MFs were significantly elevated in phenacetin treatment groups of both genders during 26- and 52-week treatments. Furthermore, sensitive to P2 interference (Spi(-)deletions) MF increased in the liver of both genders following 52-week treatment. MFs were higher after treatment for 52 weeks than after treatment for 26 weeks. Frequencies of phenacetin-induced mutations were higher in the liver than in the kidney, suggesting that the intensity of genotoxicity does not necessarily correlate with the induction of tumor formation. Results from gpt delta rat assays of repeated-dose treatments are extremely useful to elucidate the relationship between gene mutations and carcinogenesis in the target organ induced by cancer-causing agents.


Subject(s)
Analgesics, Non-Narcotic/toxicity , Hypoxanthine Phosphoribosyltransferase/genetics , Kidney/drug effects , Liver/drug effects , Phenacetin/toxicity , Point Mutation/drug effects , Animals , Cell Transformation, Neoplastic/chemically induced , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Female , Hypoxanthine Phosphoribosyltransferase/metabolism , Kidney/enzymology , Kidney/pathology , Kidney Neoplasms/chemically induced , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Liver/enzymology , Liver/pathology , Male , Mutagenicity Tests , Rats , Rats, Sprague-Dawley , Rats, Transgenic , Risk Assessment , Time Factors
6.
Toxicol Lett ; 180(3): 174-81, 2008 Aug 28.
Article in English | MEDLINE | ID: mdl-18588957

ABSTRACT

Acetaminophen (APAP) is safe at therapeutic levels but causes liver injury via N-acetyl-p-benzoquinone imine (NAPQI)-induced oxidative stress when overdose. Recent studies indicated that mitochondrial permeability transition (mPT) plays a key role in APAP-induced toxicity and leflunomide (LEF) protects against the toxicity through inhibition of c-jun NH2-terminal protein kinase (JNK)-mediated pathway of mPT. It is not clearly understood if LEF also exerts its protective effect through inhibition of APAP bioactivation to the toxic NAPQI. The present work was undertaken to study the effect of LEF on the bioactivation of APAP to NAPQI. Mechanism-based inhibition incubations performed in mouse and human liver microsomes (MLM and HLM) indicated that inhibition of APAP bioactivation to NAPQI was observed in MLM but not in HLM. Furthermore, LEF but not its active metabolite, A77-1726, was shown to be the main inhibitor. When APAP and LEF were incubated with human recombinant P450 enzymes, CYP1A2 was found to be the isozyme responsible for the inhibition of APAP bioactivation. Species variation in CYP1A2 enzymes probably accounted for the different observations in our MLM and HLM studies. We concluded that inhibition of NAPQI formation is not a probable pathway that LEF protects APAP-induced hepatotoxicity in human.


Subject(s)
Acetaminophen/antagonists & inhibitors , Acetaminophen/toxicity , Analgesics, Non-Narcotic/toxicity , Antirheumatic Agents/pharmacology , Benzoquinones/metabolism , Chemical and Drug Induced Liver Injury/prevention & control , Imines/metabolism , Isoxazoles/pharmacology , Acetaminophen/pharmacokinetics , Analgesics, Non-Narcotic/pharmacokinetics , Animals , Biotransformation , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme System/metabolism , Data Interpretation, Statistical , Glutathione/metabolism , Humans , Leflunomide , Mass Spectrometry , Mice , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Phenacetin/toxicity , Recombinant Proteins/metabolism
7.
Toxicol In Vitro ; 21(4): 734-40, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17376646

ABSTRACT

In order to elucidate the feasibility of predicting liver and kidney target-organ toxicity using in vitro cytotoxicity assay, cytotoxicity of selected chemicals, acetaminophen (AAP), mitomycin (MMC), cupric chloride (CuCl2), phenacetin, cadmium chloride (CdCl2) and aristolochic acid (AA), was studied using human hepatoma (Bel-7402) cells and human renal tubular epithelial (HK-2) cells. Cell viability and mitochondrial permeability transition (MPT) were assessed by the neutral red (NR) assay and laser scanning confocal microscope, respectively. The results of the NR assay indicated that cytotoxicity of hepatoxicants, AAP, MMC and CuCl2 in liver cells was higher than that in kidney cells. Cytotoxicitiy of nephrotoxicant, CdCl2 was lower in liver cells than that in kidney cells, but nephrotoxicant phenacetin and AA was higher cytotoxicity in liver cells than that in kidney cells. The cytotoxicity of AAP and phenacetin was strengthened in the presence of S9 mixture, indicating that they are metabolism-mediated cytotoxicants. All selected chemicals disrupted MPT in dose-dependent manner. Linear regression analysis revealed a good correlation between the IC50 values of cytotoxicity and the EC50 values of MPT in Bel-7402 cells and HK-2 cells (R2 = 0.987 and 0.823, respectively). Cytotoxicity assay in vitro using specific cells show good compatibility with target-organ toxicity in vivo. However, limitations of in vitro cytotoxicity assay are due to its incomplete process of ADME and the defect of predicting chronic toxicity effect after long-term exposure to a chemical.


Subject(s)
Chemical and Drug Induced Liver Injury/pathology , Kidney Diseases/chemically induced , Kidney Diseases/pathology , Acetaminophen/toxicity , Aristolochic Acids/toxicity , Cadmium Chloride/toxicity , Cell Line , Cell Survival/drug effects , Copper/toxicity , Humans , In Vitro Techniques , Mitochondria/drug effects , Mitochondria/ultrastructure , Phenacetin/toxicity , Regression Analysis , Subcellular Fractions/drug effects
8.
Mutat Res ; 596(1-2): 143-50, 2006 Apr 11.
Article in English | MEDLINE | ID: mdl-16464479

ABSTRACT

Chronic use of phenacetin-containing analgesics has been associated with the development of renal cancer. To establish genotoxicity as a possible cause for the carcinogenic effect of phenacetin, we exposed wild type and DNA repair deficient Xpa-/- and Xpa-/-/Trp53+/- mice (further referred as Xpa and Xpa/p53 mice, respectively), carrying a reporter lacZ gene, to 0.75% (w/w) phenacetin mixed in feed. Xpa mice completely lack the nucleotide excision repair pathway, and as such they are sensitive to some classes of genotoxic compounds. Phenacetin exposure induced a significant increase of lacZ mutations in the kidney of both Xpa and Xpa/p53 mice. A minor response was found in liver, whereas no lacZ mutation induction was observed in the spleen of these animals. Interestingly, the observed phenacetin-induced mutant frequencies were higher in male than those found in female mice. This gender difference is probably due to a difference in metabolic rate. Phenacetin-induced mutations mainly consisted of point mutations rather than deletions. The mutational spectra in the kidney of treated WT and Xpa mice were quite similar. Taken together, these results demonstrate that the human carcinogen phenacetin acts as a weak genotoxic agent in an in vivo mouse model system.


Subject(s)
Carcinogens/toxicity , Kidney/pathology , Mutagens/toxicity , Mutation , Phenacetin/toxicity , Xeroderma Pigmentosum Group A Protein/genetics , Animals , Female , Kidney/drug effects , Liver/drug effects , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Sex Characteristics , Spleen/drug effects , Spleen/pathology , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics
9.
Toxicol Pathol ; 32(2): 192-201, 2004.
Article in English | MEDLINE | ID: mdl-15200157

ABSTRACT

As part of the international evaluation program coordinated by ILSI/HESI, the potential of DNA repair deficient Xpa-/- mice and the double knockout Xpa-/-.p53+/- mice for short term carcinogenicity assays was evaluated. For comparison also wild-type C57BL/6 mice (WT) were included in these studies. Four test compounds were administered to groups of 15 male and 15 female Xpa-/- mice, Xpa-/-.p53+/- mice and WT mice for 39 weeks. The model compounds investigated were haloperidol, reserpine (nongenotoxic rodent carcinogens, putative human noncarcinogens), phenacetin (genotoxic rodent carcinogen, suspected human carcinogen), and D-mannitol (noncarcinogen in rodents and humans). The test compounds were administered as admixture to rodent diet at levels up to 25 mg/kg diet for haloperidol, 7.5 mg/kg diet for reserpine, 0.75% for phenacetin, and 10% for D-mannitol. These levels included the maximum tolerable dose (MTD). Survival was not affected with any of the test compounds. Haloperidol, reserpine and D-mannitol were negative in the carcinogenicity assay with Xpa-/- and Xpa-/-.p53+/- mice, showing low and comparable tumor incidences in controls and high-dose animals. The results obtained with phenacetin may be designated equivocal in Xpa-/-.p53+/- mice, based on the occurrence of a single rare tumor in the target organ (kidney) accompanied by a low incidence of hyperplastic renal lesions and a high incidence of karyomegaly. These results are in agreement with the currently known carcinogenic potential of the 4 test compounds in humans.


Subject(s)
Carcinogenicity Tests/methods , Haloperidol/toxicity , Mannitol/toxicity , Neoplasms, Experimental/chemically induced , Phenacetin/toxicity , Reserpine/toxicity , Administration, Oral , Animals , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Diet , Disease Models, Animal , Dose-Response Relationship, Drug , Haloperidol/administration & dosage , Mannitol/administration & dosage , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neoplasms, Experimental/pathology , Phenacetin/administration & dosage , Reproducibility of Results , Reserpine/administration & dosage , Time Factors , Xeroderma Pigmentosum Group A Protein
13.
Drug Chem Toxicol ; 24(2): 117-27, 2001 May.
Article in English | MEDLINE | ID: mdl-11360430

ABSTRACT

The developmental toxicities of five test compounds including carbon tetrachloride, urethane, phenacetin, parathion, and chloroform, were evaluated using Frog Embryo Teratogenesis Assay--Xenopus (FETAX), with minor modification. Post-isolation mixtures of differently-induced rat liver microsomes (phenobarbital- (PB), beta-naphthoflavone- (beta-NF), and isoniazid- (INH)-induced preparations) were co-cultured directly with X. laevis embryos. Results from these studies suggest that the Aroclor 1254-induced MAS could effectively be replaced by a mixed lot of PB-, beta-NF-, and INH-induced rat liver microsomes. Each of the test materials were found to be developmentally toxic when bioactivated by the mixed MAS.


Subject(s)
Biotransformation , Embryo, Mammalian/drug effects , Embryo, Nonmammalian , Toxicity Tests , Xenopus laevis/embryology , Abnormalities, Drug-Induced/etiology , Animals , Carbon Tetrachloride/toxicity , Chloroform/toxicity , Coculture Techniques , Isoniazid , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Parathion/toxicity , Phenacetin/toxicity , Phenobarbital , Rats , Urethane/toxicity , beta-Naphthoflavone
14.
Toxicol Appl Pharmacol ; 161(2): 153-9, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-10581209

ABSTRACT

Five chemicals, known to induce kidney tumors in rats, were assayed for their ability to induce DNA damage and formation of micronuclei in primary cultures of rat and human kidney cells and in the kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, and of micronuclei frequency were obtained in primary kidney cells from both rats and humans with the following concentrations of the five test compounds: lead acetate (not tested for micronuclei induction) and potassium bromate from 0.56 to 1.8 mM, phenacetin from 1 to 3.2 mM, and 1, 4-dichlorobenzene and nitrilotriacetic acid from 1.8 to 5.6 mM. In terms of DNA-damaging potency all the five chemicals were more active in rat than in human kidney cells, whereas the potencies in inducing micronuclei formation were similar in the two species with the exception of 1,4-dichlorobenzene, which was slightly more potent in human than in rat cells. Consistently with the results observed in vitro, statistically significant increases in the average frequency of both DNA breaks and micronucleated cells were detected in the kidney of rats given po a single (12 LD50) or three successive daily doses (13 LD50) of the five test compounds. 4, 4'-Methylenedianiline, a carcinogen which does not induce kidney tumors in rats, gave negative responses in both in vitro and in vivo assays. These findings give evidence that kidney carcinogens may be identified by short-term genotoxicity assays using as target kidney cells and show that the five chemicals tested produce in primary cultures of kidney cells from human donors effects similar to those observed in rats.


Subject(s)
Carcinogens/toxicity , DNA Fragmentation/drug effects , Kidney/drug effects , Adult , Aniline Compounds/toxicity , Animals , Bromates/toxicity , Cells, Cultured , Chlorobenzenes/toxicity , Comet Assay , Dose-Response Relationship, Drug , Female , Humans , Kidney/cytology , Male , Micronucleus Tests , Middle Aged , Nitrilotriacetic Acid/toxicity , Organometallic Compounds/toxicity , Phenacetin/toxicity , Rats , Rats, Sprague-Dawley , Species Specificity
15.
Toxicol Sci ; 50(1): 82-9, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10445756

ABSTRACT

The mechanisms underlying phenacetin-induced toxicity and carcinogenicity are not clear. In particular, it is not known whether these effects are mediated by metabolic activation of the drug. CYP1A2 is known to metabolize phenacetin in vitro. To determine the role of this enzyme in vivo, the toxicity and carcinogenicity of phenacetin was examined in Cyp1a2-null mice (that lack CYP1A2). Six- to 8-week-old wild type (+/+) or null (-/-) mice were fed either a control diet, or one containing 1.25% phenacetin, ad libitum for up to 67 weeks. Representative groups of mice were examined for phenacetin-induced toxicity and carcinogenicity after 36, 48, 58, or 67 weeks of feeding. Consistent with the known role of CYP1A2 in phenacetin metabolism, plasma levels of phenacetin were higher and acetaminophen levels lower in the (-/-) mice fed phenacetin compared to phenacetin-fed (+/+) controls. Weight gain was significantly depressed in both groups of phenacetin-fed mice after 4 weeks of feeding, and continued to be lower for the remainder of the experiment, compared to controls. Hepatomegaly and splenomegaly were more severe in (-/-) mice but present in both genotypes fed phenacetin at all time points assessed. Histological analysis of liver, kidney, spleen, and urogenital tract also revealed a differential response in the (-/-) mice fed phenacetin compared to (+/+) mice fed the same diet. Further, mortality was the most severe in the (-/-) mice fed phenacetin than in all other groups. Despite significant toxicity in (-/-) mice fed phenacetin, only one renal carcinoma was found among them. Results from this work demonstrate that, in the absence of CYP1A2, phenacetin is more toxic than in controls. This provides evidence that metabolism of phenacetin by CYP1A2 alters toxicity in vivo, and suggests that alternate CYP1A2-independent metabolic pathways contribute to its toxicity.


Subject(s)
Acetaminophen/toxicity , Carcinogens/toxicity , Cytochrome P-450 CYP1A2/physiology , Phenacetin/toxicity , Acetaminophen/blood , Animals , Carcinoma/chemically induced , Female , Kidney Neoplasms/chemically induced , Male , Mice , Organ Size/drug effects , Phenacetin/administration & dosage , Phenacetin/blood , Survival Rate , Time Factors , Urinary Bladder/drug effects , Urinary Bladder/pathology
17.
Regul Toxicol Pharmacol ; 28(3): 226-9, 1998 Dec.
Article in English | MEDLINE | ID: mdl-10049794

ABSTRACT

A systemic exposure-based alternative to the MTD (maximally tolerated dose) for high-dose selection in carcinogenicity studies of pharmaceuticals has been accepted by the ICH (International Conference on Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use). As a result of a retrospective analysis performed by the U.S. FDA (United States Food and Drug Administration), a rat/human relative systemic exposure ratio of 25 is proposed by the ICH as an acceptable pharmacokinetic endpoint for high-dose selection. For use as a dose selection criterion, it is particularly important that the magnitude of the relative systemic exposure ratio should be sufficient to detect human pharmaceuticals classified by IARC (International Agency for Research on Cancer, World Health Organization) as known (category 1) or probable (category 2A) human carcinogens. For one of these, phenacetin (an IARC 2A compound and a rat carcinogen), a systemic exposure ratio of 15 was calculated by the FDA. This calculation was based on a number of extrapolations. The present study reports the actual systemic exposure to phenacetin in the rat under conditions mimicking the conditions in the carcinogenicity study used by the FDA to calculate the relative systemic exposure ratio of 15. The ratio was found to be 7, indicating that the carcinogenic potential of this particular probable human carcinogen could be detected at a considerably lower systemic exposure ratio than that proposed by the ICH.


Subject(s)
Carcinogenicity Tests , Carcinogens/toxicity , Phenacetin/toxicity , Animals , Area Under Curve , Dose-Response Relationship, Drug , Eating , Half-Life , Humans , Male , Phenacetin/blood , Rats , Rats, Sprague-Dawley , Retrospective Studies
18.
Mutat Res ; 391(3): 215-31, 1997 Jul 14.
Article in English | MEDLINE | ID: mdl-9268047

ABSTRACT

Recently, we designed a fast and simple method to obtain nuclei for the alkaline SCG assay and we tested it with mouse liver, lung, kidney, spleen, and bone marrow. Instead of isolating organ cells by trypsinization, we homogenized tissue and isolated the nuclei. Each organ was minced, and the mince was suspended in chilled homogenizing buffer containing NaCl and Na2EDTA, homogenized gently using a Potter-type homogenizer set in ice, and then centrifuged. The nuclei from the precipitate were used for the assay. To evaluate the validity of this method, we tested the genotoxicity in mouse organs of 11 chemical mutagens with different modes of action. Mice were sacrificed 3 and 24 h after administration of each mutagen. Treatment with three alkylating agents (MMS, EMS, and MNNG), a DNA crosslinking agent (MMC), two aromatic amines (2-AAF and phenacetin), a polycyclic aromatic hydrocarbon (B[a]P), and two inorganic chemicals (KBrO3 and K2CrO4) increased migration of the DNA from mouse organs. 5-FU (a base analog) and colchicine (a spindle poison) treatment produced negative results in all organ studied. Considering that the alkaline SCG assay detects genotoxicity as DNA fragments derived from DNA single-strand breaks and alkali-labile damage, our results showed that the SCG assay using our homogenization technique detected chemical mutagens as a function of their modes of action.


Subject(s)
Electrophoresis/methods , Mutagenicity Tests/methods , Mutagens/toxicity , Alkylating Agents/toxicity , Animals , Bone Marrow/drug effects , Colchicine/toxicity , Cross-Linking Reagents/toxicity , Fluorouracil/toxicity , Kidney/drug effects , Liver/drug effects , Lung/drug effects , Male , Mice , Mice, Inbred Strains , Mitomycin/toxicity , Phenacetin/toxicity , Polycyclic Aromatic Hydrocarbons/toxicity , Spleen/drug effects
19.
Ukr Biokhim Zh (1978) ; 69(3): 72-6, 1997.
Article in Russian | MEDLINE | ID: mdl-9505365

ABSTRACT

Effects of amidopyrine, phenacetin and paracetamol on the viability and energetic state of isolated rat hepatocytes were compared. During incubation in minimal salt solution all drugs in concentrations above 1 mM in dose-dependent manner decreased the viability of hepatocyte suspension assessed by trypan blue dye inclusion and inhibited the ATP synthesis and the respiratory activity. Cytotoxic effect of chemicals decrease in the order: amidopyrine-->phenacetin-->paracetamol. The inhibition of the rate of endogenous respiration were accompanied by stimulation of oxygen consumption in nonmitochondrial systems. An uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, and disruption of plasma membrane by digitonin followed by substrate succinate addition did not restore the respiratory activity of hepatocytes up to the level of control cells. These data show that cytotoxicity of the chemicals is determined by their interaction with enzymes of mitochondrial respiratory chain.


Subject(s)
Acetaminophen/toxicity , Aminopyrine/toxicity , Analgesics, Non-Narcotic/toxicity , Anti-Inflammatory Agents, Non-Steroidal/toxicity , Mitochondria, Liver/drug effects , Phenacetin/toxicity , Adenosine Triphosphate/metabolism , Animals , Cell Respiration/drug effects , Energy Metabolism/drug effects , In Vitro Techniques , Oxygen Consumption/drug effects , Rats , Rats, Wistar
20.
Mutat Res ; 347(2): 73-8, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7651467

ABSTRACT

To examine the suitability of using rat peripheral blood from animals used in subchronic toxicity studies for micronucleus analysis, we orally administered phenacetin or 6-mercaptopurine for 14 days to groups of six rats and compared their micronucleus frequencies to the bone marrow micronucleus frequencies of rats similarly treated for only 2 days. In the 14-day test, phenacetin significantly increased the frequency of micronucleated reticulocytes in peripheral blood at 500 mg/kg starting from day 9, and at 750 and 1500 mg/kg starting from day 6; 6-mercaptopurine gave a positive response at 20 mg/kg starting from day 6. Positive responses in the bone marrow assay were obtained at the same dose levels. In the 2-day test, micronucleated polychromatic erythrocyte frequencies increased significantly at 1000 and 2000 mg/kg for phenacetin, and at 50, 100, and 200 mg/kg for 6-mercaptopurine. These results suggest that micronucleus assays using peripheral blood from rats in subchronic animal studies of phenacetin and 6-mercaptopurine are feasible and at least as sensitive for the assessment of micronuclei as an acute bone marrow micronucleus test.


Subject(s)
Erythrocytes/drug effects , Mutagens/toxicity , Animals , Dose-Response Relationship, Drug , Erythrocytes/ultrastructure , Male , Mercaptopurine/toxicity , Micronucleus Tests/methods , Phenacetin/toxicity , Rats , Rats, Sprague-Dawley
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