Theoretical investigation on the contribution of HO, SO4- and CO3- radicals to the degradation of phenacetin in water: Mechanisms, kinetics, and toxicity evaluation.

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.

No obvious toxicological influences of 50 µL microsampling from rats administered phenacetin as a drug with hematological toxicity.

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.

Bio-isosteric replacement of amide group with 1,2,3-triazole in phenacetin improves the toxicology and efficacy of phenacetin-triazole conjugates (PhTCs).

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.

Prevention of acetaminophen (APAP)-induced hepatotoxicity by leflunomide via inhibition of APAP biotransformation to N-acetyl-p-benzoquinone imine.

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.

In vitro cytotoxicity assay with selected chemicals using human cells to predict target-organ toxicity of liver and kidney.

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.

Phenacetin acts as a weak genotoxic compound preferentially in the kidney of DNA repair deficient Xpa mice.

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.

Carcinogenicity of bucetin in (C57BL/6 X C3H)F1 mice.

The carcinogenicity of bucetin [(3-hydroxy-p-butyrophenetidide) CAS: 1083-57-4], an antipyretic analgesic drug, was examined in 300 (C57BL/6 X C3H)F1 mice. Groups of 50 mice of each sex were treated with 1.5 or 0.75% bucetin in their basal diet for 76 weeks and then fed a basal diet for 8 weeks. Control groups were given a basal diet for 84 weeks. In 10 of 46 (22%) male mice given the high dose of bucetin and in 6 of 45 (13%) given the low dose, renal cell tumors were induced. Dysplastic lesions of the proximal tubules were frequently seen in the males given bucetin in a dose-related fashion. Neither tumorous nor preneoplastic lesions developed in the kidneys of bucetin-treated female mice and control animals. Papilloma of the urinary bladder in 1 male mouse and papillary or nodular hyperplasia in 9 mice of both sexes were observed in groups given the high dose of bucetin.

Induction of renal pelvic carcinoma by phenacetin in hydronephrosis-bearing rats of the SD/cShi strain.

Carcinogenicity of phenacetin (PH) to the urinary tract was tested with the use of spontaneously hydronephrosis-bearing rats. In Experiment 1, 55 SD/cShi male rats were fed with 2% PH-containing diet for 85 weeks, and 32 SD/cShi male rats fed basal diet for 85 weeks served as controls. Forty-three of 53 rats fed with PH had renal pelvic carcinoma with lung metastases in three. The mean induction time was 78 weeks. Ureteral carcinoma and urinary bladder carcinoma were observed in 2 and 6 of 53 rats given PH, respectively. No urinary tract carcinoma was found in control animals. In Experiment 2, early lesions of the kidney affected by PH were also evaluated with the use of SD/cShi and Sprague-Dawley (SD) rats. Two groups of animals containing 6 SD/cShi or 6 SD male rats per group were fed 2% PH-containing diet for 8 weeks. Control animals containing 6 SD/cShi rats or 6 SD rats were fed basal diet for 8 weeks. Simple hyperplasia was found in 5 of 6 SD/cShi rats given PH and 2 of 6 SD/cShi control rats. Papillary necrosis was seen in 4 of 6 SD/cShi and 2 of 6 SD rats given PH. SD/cShi rats, especially those treated with PH, showed higher but not significant 5-bromo-2'-deoxyuridine labeling indices in the covering epithelium of the renal pelvis and papillae. In this short term experiment PH and its metabolites, N-hydroxyphenacetin and N-acetyl-p-aminophenol, were measured in urine and plasma by using high performance liquid chromatography. Significantly higher PH and slightly higher metabolites were detected in urine and plasma of SD/cShi rats compared to SD rats. These results indicated that the renal pelvis of SD/cShi rats had more sensitivity to PH carcinogenicity. This paper provides experimental proof of PH carcinogenicity toward the renal pelvis in an animal model.

Activation of N-hydroxyphenacetin to mutagenic and nucleic acid-binding metabolites by acyltransfer, deacylation, and sulfate conjugation.

N-Hydroxyphenacetin was activated to a mutagen in the Salmonella-Ames test by rabbit liver acyltransferase, rat liver cytosol, and rat liver microsomes. N-[ring]3H]-Hydroxyphenacetin was bound to transfer RNA when activated by acyltransferase from rabbit or rat liver or rat liver microsomes. The acyltransferase-catalyzed binding was not inhibited by paraoxon, a deacetylase inhibitor. The use of N-hydroxyphenacetin radioactively labeled in the acetyl group, as well as the ring, indicated that deacetylation was involved in the microsome-catalyzed binding reaction. In addition, the microsome-catalyzed binding was inhibited 90% by paraoxon. p-Nitrosophenetole, a deacetylated derivative of N-hydroxyphenacetin, was synthesized and bound to transfer RNA without enzymatic activation. Activation of N-hydroxyphenacetin by sulfate conjugation was also found to lead to binding to transfer RNA. The data implicated acyl transfer, deacetylation, and sulfate conjugation as possible routes for the activation of N-hydroxyphenacetin.

Metabolic activation and genotoxicity of N-hydroxy-2-acetylaminofluorene and N-hydroxyphenacetin derivatives in Reuber (H4-II-E) hepatoma cells.

Derivatives of both N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxyphenacetin (N-OH-P) were tested for their ability to cause DNA damage in Reuber (H4-II-E) cells using the alkaline elution technique. Reuber cells are devoid of N-OH-AAF deacylase, N,O-acyltransferase, and sulfotransferase activities. The hydroxamic acids themselves caused very little DNA damage, while N-hydroxy-2-aminofluorine (20 to 100 microM), N-hydroxyphenetidine (20 to 200 microM), and p-nitrosophenetole (10 to 100 microM) all caused dose-dependent damage. The dose-dependent DNA damage caused by N-acetoxy-2-acetylaminofluorene (5 to 25 microM) was completely inhibited by the deacylase inhibitor paraoxon (100 microM). In the presence of both partially purified rabbit liver cytosolic N,O-acyltransferase and guinea pig liver microsomal deacylase, N-OH-AAF was genotoxic. Neither paraoxon nor tRNA had any effect on the DNA damage induced by N-OH-AAF in the presence of N,O-acyltransferase, while paraoxon completely inhibited the damage when N-OH-AAF was incubated in the presence of guinea pig deacylase, and N-OH-P only caused slight DNA damage at higher concentrations of enzyme. In addition, partially purified guinea pig liver deacylase and N-OH-AAF (25 microM) caused 2600 revertants in the Salmonella test system, while only 380 revertants were seen with a 40-fold greater concentration of N-OH-P (1000 microM). The mutagenicity of both N-OH-AAF and N-OH-P was completely inhibited by paraoxon. Thus, it is clear that metabolites of N-OH-AAF formed outside the cell are capable of passing both the cellular and nuclear membranes to cause genotoxicity. Metabolic activation of N-OH-AAF by either the membrane-bound deacylase or the cytosolic N,O-acyltransferase caused genotoxicity via a deacetylation process. Metabolic activation of N-OH-P by guinea pig deacylase caused low levels of DNA damage, whereas activation by N,O-acyltransferase was not sufficient to cause genotoxicity.

Effect of phenacetin and N-alkylacetanilides on N-2-fluorenylacetamide hepatocarcinogenesis.

Phenacetin, N-methylacetanilide and N-ethylacetanilide prevented N-2-fluorenylacetamide hepatocarcinogenesis in F344 strain rats. Phenacetin was most effective followed by N-ethylacetanilide. Phenacetin was not carcinogenic when fed at 0.8% level for 16 weeks followed by control diet for another 10 weeks.

Reactive metabolites of phenacetin and acetaminophen: a review.

Phenacetin can be metabolized to reactive metabolites by a variety of mechanisms. (1) Phenacetin can be N-hydroxylated, and the resulting N-hydroxyphenacetin can be sulfated or glucuronidated. Whereas phenacetin N-O sulfate immediately rearranges to form a reactive metabolite which may covalently bind to protein, phenacetin N-O glucuronide slowly rearranges to form reactive metabolites. Incubation of the purified phenacetin N-O glucuronide under a variety of conditions suggests that N-acetyl-p-benzoquinone imine is a reactive metabolite. This metabolite covalently binds to protein, reacts with glutathione to form an acetaminophen-glutathione conjugate, is reduced by ascorbate to acetaminophen or is partially hydrolyzed to acetamide. (2) Phenacetin can be O-deethylated to acetaminophen, and acetaminophen can be converted directly to a reactive metabolite which may be also N-acetyl-p-benzoquinone imine. (3) Phenacetin can be sequentially N-hydroxylated and O-deethylated to N-hydroxyacetaminophen which spontaneously dehydrates to N-acetyl-p-benzoquinone imine. (4) Phenacetin can be 3, 4-epoxidated to form an alkylating and an arylating metabolite. In the presence of glutathione, a S-ethylglutathione conjugate and an acetaminophen-glutathione conjugate are formed. In the absence of glutathione, the alkylating metabolite may bind to protein and the arylating metabolite is completely hydrolyzed to acetamide and another arylating metabolite which may bind to protein. The structures of the alkylating and arylating metabolites are unknown. Control experiments have shown that in pathway (1) the phenolic oxygen of the acetaminophenglutathione conjugate is derived from water, whereas in pathways (2) and (3) the phenolic oxygen of this metabolite is derived from phenacetin. In pathway (4) the phenolic oxygen was 50% derived from molecular oxygen and 50% from phenacetin. Administration of [p-(18)0]phenacetin to hamsters revealed only a 10% loss of (18)0 in the acetaminophen mercapturic acid (the further metabolic product of the glutathione conjugate) which suggests that, in the hamster, pathways (2) and/or (3) are the primary mechanism of conversion of phenacetin to reactive metabolites in vivo.

Role of CYP1A2 in the toxicity of long-term phenacetin feeding in mice.

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.

Thymosin fraction 5 does not influence urinary tract carcinogenesis by phenacetin and N-butyl-N-(4-hydroxybutyl)nitrosamine in NON/Shi mice.

The effect of thymosin fraction 5 (TF5) on the promotion and progression phases of urinary tract carcinogenesis induced by consecutive administration of phenacetin and N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in NON/Shi mice was investigated. The study was carried out twice with a minor modification to the protocol in the second experiment. Fifty-seven male NON/Shi mice in experiment 1 and 100 mice in experiment 2 were each divided into four groups. Phenacetin was administered for 8 weeks in experiment 1 and 12 weeks in experiment 2, and subsequently BBN was given for 6 weeks in both cases, for total observation periods of 30 and 34 weeks, respectively. Sixty micrograms of TF5 per mouse was inoculated subcutaneously twice a week during (group 2) or after (group 3) BBN exposure, or both periods (group 4). Group 1 served as a control group without TF5 treatment. Histopathological examination revealed no effects on either induction of urinary tract carcinomas or distant metastasis from renal pelvic carcinomas in either experiment.

Amphibian organ culture in experimental toxicology: the effects of paracetamol and phenacetin on cultured tissues from urodele and anuran amphibians.

Organ cultures of various tissues from urodele amphibians deacetylate paracetamol to p-aminophenol, which polymerises to form a brown precipitate. Paracetamol addition results in a loss of glycogen and lactate dehydrogenase (LDH) from urodele liver cultures and an increase in glucose release, and in LDH loss from kidney cultures. Organ cultures from anuran amphibians are unable to metabolise paracetamol and are not affected by its presence in the culture medium. The addition of unpolymerised p-aminophenol resulted in a loss of LDH from urodele and anuran organ cultures, whilst the addition of polymerised p-aminophenol had no such effects. This suggests that the toxic effects which follow the addition of paracetamol to urodele organ cultures are caused by unpolymerised p-aminophenol, a known toxicant in mammals. Cultures from both urodele and anuran amphibians are able to deacetylate phenacetin to p-phenetidine, but p-phenetidine was found to be much less toxic to amphibian tissues than p-aminophenol, causing LDH loss from kidney cultures only at very high dose levels.

Neoplasia in the rat induced by N-hydroxyphenacetin, a metabolite of phenacetin.

N-hydroxyphenacetin, a phenacetin metabolite, was fed to rats as a 0.05-0.5% dietary supplement. After 9 months, tumours of the liver were found in 36 of 64 animals. One animal also developed a renal tumour. No tumours were found in control animals. The findings implicate phenacetin as a carcinogen and suggest that N-hydroxyphenacetin may be the metabolite responsible.

Hepatotoxicity of phenacetin and paracetamol in the Gunn rat.

Both phenacetin and paracetamol produce acute centrilobular liver necrosis in the homozygous Gunn rat. Paracetamol is more hepatotoxic than phenacetin, and both are more hepatotoxic to the homozygous Gunn rat than to the heterozygous Gunn rat or to the albino rat. These findings have relevance to the role of the compounds in the clinical syndromes of paracetamol induced liver necrosis and analgesic nephropathy.

Toxicological screening models: drug-induced oxidative hemolysis.

In an attempt to obtain a simple screening system for the assessment of toxic-hemolytic effects of chemical substances, a battery of hematological tests was used. Phenacetin served as reference substance. The drug caused reversible formation of methemoglobin and Heinz bodies and an increase in peripheral reticulocytes after 2 and 4 weeks of treatment. Furthermore, an increase in the mean corpuscular volume of red blood cells (RBC) and the volume of RBC ghosts in hypotonic solutions, and a decrease of the mean corpuscular fragility was observed. The latter changes are considered to be a consequence of regenerative RBC compensation rather than due to structural membrane alteration caused by the drug. The results suggest that only a combination of several hematological tests can provide comprehensive information about the hemolytic potential of chemical substances, and that for screening purposes small numbers of animals are often sufficient.