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The Ames test is used worldwide to initially screen the mutagenic potential of new chemicals. In the standard Ames test, S. typhimurium strains (TA100, TA98, TA1535, and TA1537) and Escherichia coli (WP2uvrA) are treated with substances with/without cytochrome P450s (CYPs)-induced rat S9 fractions for identifying mutagens and pro-mutagens. However, many substances show completely different toxicity patterns depending on whether the liver S9 fraction belongs to rats or humans. The natural product Polygoni Multiflori Radix (PMR) can also show bacterial reverse mutation, followed by the rat or human liver S9 fraction. While PMR elicits reverse mutations in the TA1537 strain in rat liver S9 but not in human liver S9, this mechanism has not been verified yet. To explain this, the differences in metabolic enzymes compositions commonly observed between rats and humans have been implicated. This study aimed to explore the key factors that cause differences in the genotoxicity of PMR between rat and human liver S9 metabolic enzymes. The results of next-generation sequencing (NGS) analysis showed that both rat and human metabolic enzymes caused similar mutations in TA1537. However, when the metabolic enzymes in each S9 fraction were analyzed using ion mobility tandem mass spectrometry (IM-MS), rat- and human-specific enzymes were identified among the cytochrome (CYP) family, especially aryl hydrocarbon receptor (AHR)-related CYPs. These findings suggest that CYP1A1 isoforms contribute to the mechanism of PMR in the Ames test. Therefore, an in vitro Ames test might be more reliable in predicting genotoxicity for both rodents and humans. This will also help overcome the limitations of laboratory animal-based toxicity evaluations, which provide unreliable results due to interspecies differences between humans and rodents.

Asunto(s)

Pruebas de Mutagenicidad , Mutágenos , Salmonella typhimurium , Animales , Humanos , Pruebas de Mutagenicidad/métodos , Ratas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Mutágenos/toxicidad , Hígado/efectos de los fármacos , Hígado/metabolismo , Activación Metabólica , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Mutación , Daño del ADN/efectos de los fármacos , Fallopia multiflora/química , Masculino

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Among all the heavy metals, Pb, Cd, and As are the most harmful pollutants in the environment. They reach into the organisms via various levels of food chains i.e. air and water. Glutathione-s-transferase (GST, E.C. 2.5.1.18), a key enzyme of xenobiotics metabolism, plays an important role in the removal of several toxicants. The present study aimed to evaluate any inhibitory action of these heavy metals on the GST enzyme isolated from the hepatic tissues of rats. A 10 % (w/v) homogenate of rat liver was prepared in cold and centrifuged at 4 °C at 9000xg for 30 min. The supernatant was collected and kept frozen at -20 °C or used fresh for carrying out different experiments. The activity of GST was monitored spectrophotometrically at 340 nm using 220 µg of soluble protein with varying equal substrate concentrations (0.125-2 mM) in phosphate buffer (50 mM, pH 6.5). To assess the impact of heavy metals on the enzyme activity, different concentrations of Cd (0-0.6 mM) and Pb (0-2 mM) were added to the reaction mixture followed by monitoring the residual activity. The optimum temperature and pH of rat liver GST were found to be 37 °C and 6.5, respectively. The Km value for GST was 0.69 mM and the Vmax was found to be 78.67 U/mg. The Cd and Pb significantly altered the kinetic behaviour of the enzyme. The Vmax and Kcat/Km parameters of GST were recorded to be decreased after interaction with Cd and Pb individually and showed a mixed type of inhibition pattern suggesting that these inhibitors may have a greater binding affinity either for the free enzyme or the substrate-enzyme complex. These metals showed a time-dependent enzyme inhibition profile. Cd was found to be the most potent inhibitor when compared to other treated metals; the order of inhibitory effect of metal ions was Cd>Pb>As. The in silico ion docking analysis for determining the probable interactions of Cd and Pb with fragmented GST validated that Cd exhibited higher inhibition potential for the enzyme as compared to Pb. The results of the present study indicated that exposure of both the Cd and Pb may cause significant inhibition of hepatic GST; the former with higher inhibitory potential than the later. However, As proved to be least effective against the enzyme under the aforesaid experimental conditions.

RESUMEN

1. Isolated perfused rat liver (IPRL) experiments have been used to answer clearance-related questions, including evaluating the impact of pathological and physiological processes on hepatic clearance (CLH). However, to date, IPRL data has not been evaluated for in vivo CLH prediction accuracy.2. In addition to a detailed overview of available IPRL literature, we present an in-depth analysis of the performance of IPRL in CLH prediction.3. While the entire dataset poorly predicted CLH (GAFE = 3.2; 64% within 3-fold), IPRL conducted under optimal experimental conditions, such as in the presence of plasma proteins and with a perfusion rate within 2-fold of physiological liver blood flow and corrected for unbound fraction in the presence of red blood cells, can accurately predict rat CLH (GAFE = 2.0; 78% within 3-fold). Careful consideration of experimental conditions is needed to allow proper data analysis.4. Further, isolated perfused liver experiments in other species, including human livers, may allow us to address the current in vitro-in vivo disconnects of hepatic metabolic clearance and improve our methodology for CLH predictions.

RESUMEN

Kokusaginine is a bioactive ingredient extracted from Ruta graveolens L., which has a range of biological activities. Its pharmacokinetic (PK) properties are particularly important for clinical applications; however, they have not been fully elucidated. In addition, the effect of sex differences on drug metabolism is increasingly being recognized, but most studies have ignored this important factor. This study aims to fill this knowledge gap by taking an in-depth look at the PK properties of kokusaginine and how gender affects its metabolism and distribution in the body. It also lays the foundation for clinical drug development. In this study, a sensitive ultra-high-performance liquid chromatography (UPLC) method was developed and validated for quantifying kokusaginine in Sprague Dawley (SD) rat plasma and tissue homogenates. Metabolic stability was evaluated in vitro using gender-specific liver microsomes. Innovatively, we incorporated sex as a variable into both in vitro and in vivo PK studies in SD rats, analyzing key parameters with Phoenix 8.3.5 software. The developed UPLC method demonstrated high sensitivity and precision, essential for PK analysis. Notably, in vitro studies revealed a pronounced sex-dependent metabolic variability (p < 0.05). In vivo, gender significantly affected the Area Under the Moment Curve (AUMC)(0-∞) of the plasma PK parameter (p < 0.05) and the AUMC(0-t) of brain tissue (p < 0.0001), underscoring the necessity of sex-specific PK assessments. The calculated absolute bioavailability of 71.13 ± 12.75% confirmed the favorable oral absorption of kokusaginine. Additionally, our innovative tissue-plasma partition coefficient (Kp) analysis highlighted a rapid and uniform tissue distribution pattern. This study presents a sex-inclusive PK evaluation of kokusaginine, offering novel insights into its metabolic profile and distribution. These findings are instrumental for informing clinical medication practices, dosage optimization, and a nuanced understanding of drug efficacy and safety in a sex-specific context.

RESUMEN

Introduction: The presence of phytochemicals in herbal medicines can lead to herb-drug interactions, altering the levels of these compounds and conventional drugs in the bloodstream by influencing CYP450 activity. Considering curcumin's effect on the CYP enzymes responsible for tramadol metabolism, it is essential to assess the potential interaction between curcumin and tramadol when administered together. Materials and methods: The pharmacokinetics of tramadol were examined in rats receiving either single or multiple doses of curcumin (80 mg/kg) compared to rats without curcumin treatment. Tramadol liver perfusion was conducted on all rat groups and perfusate samples were collected at specified intervals. Tramadol and its main metabolite were detected using an HPLC system coupled with a fluorescence detector. Results: Tramadol concentrations were notably higher in the co-administered group compared to both the control and treatment groups. Conversely, lower concentrations of M1 were observed in the co-administered and treatment groups compared to the control group. The AUC0-60 parameters for tramadol were as follows: 32944.8 ± 1355.5, 22925.7 ± 1650.1, and 36548.0 ± 2808.4 ng⋅min/ml for the control, treatment, and co-administered groups, respectively. Both the co-administered and treatment groups exhibited a lower AUC0-60 of M1 compared to the control group. The lack of significant difference in Cmax and AUC0-60 of M1 between the treatment and co-administered groups suggests that single and multiple doses of curcumin have comparable effects on CYP2D6. Conclusions: These results indicate a potential for drug interactions when curcumin and tramadol are taken together. Furthermore, the influence of curcumin on tramadol metabolism varied between single and multiple oral administrations of curcumin. Hence, it is vital to highlight this interaction in clinical settings and conduct additional research to fully understand the clinical implications of combining curcumin and tramadol.

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BACKGROUND: Our recent studies showed that prolonged administration of novel atypical antipsychotics affected the expression and activity of cytochrome P450 (CYP), as demonstrated in vitro on human hepatocytes and in vivo on the rat liver. The aim of the present work was to study the effect of repeated treatment with asenapine, iloperidone, and lurasidone on the expression of transcription factors regulating CYP drug-metabolizing enzymes in rat liver. METHODS: The hepatic mRNA (qRT-PCR) and protein levels (Western blotting) of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor (PPARγ) were measured in male Wistar rats after 2 week-treatment with asenapine, iloperidone or lurasidone. RESULTS: The 2-week treatment with asenapine significantly diminished the AhR and PXR expression (mRNA, protein level), and CAR mRNA level in rat liver. Iloperidone lowered the AhR and CAR expression and PXR protein level. Lurasidone did not affect the expression of AhR and CAR, but increased PXR expression. The antipsychotics did not affect PPARγ. CONCLUSIONS: Prolonged treatment with asenapine, iloperidone, or lurasidone affects the expression of transcription factors regulating the CYP drug-metabolizing enzymes. The changes in the expression of AhR, CAR, and PXR mostly correlate with alterations in the expression and activity of respective CYP enzymes found in our previous studies. Since these transcription factors are also engaged in the expression of phase II drug metabolism and drug transporters, changes in their expression may affect the metabolism of endogenous substrates and pharmacokinetics of concomitantly used drugs.

Asunto(s)

Antipsicóticos , Sistema Enzimático del Citocromo P-450 , Compuestos Heterocíclicos de 4 o más Anillos , Isoxazoles , Hígado , Clorhidrato de Lurasidona , Receptor X de Pregnano , Ratas Wistar , Receptores Citoplasmáticos y Nucleares , Animales , Antipsicóticos/farmacología , Masculino , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Ratas , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptor X de Pregnano/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Clorhidrato de Lurasidona/farmacología , Isoxazoles/farmacología , Piperidinas/farmacología , Receptor de Androstano Constitutivo/metabolismo , Dibenzocicloheptenos/farmacología , Receptores de Esteroides/metabolismo , PPAR gamma/metabolismo , Factores de Transcripción/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética

RESUMEN

Arsenic trioxide (ATO), one of the oldest and most frequently used poisons, is well-known in forensic science for inducing hepatotoxicity. The regulation of peroxisomal antioxidative enzyme catalase (CAT) involves intricate mechanisms at both transcriptional and post-transcriptional levels. However, the molecular mechanisms underlying the regulation of CAT gene expression in hepatic cells remain elusive. Furthermore, the regulation of CAT gene expression evident in animals administered with ATO in vivo is not well-explored, although several studies have revealed ATO-induced reductions in CAT enzymatic activity in rat livers. In this study, we revealed ATO-dependent reductions in CAT gene expression in both rat liver and Huh-7 human hepatoma cells. Our results indicate that the decline in CAT enzymatic activity can be attributed, at least in part, to the downregulation of its gene expression. The ATO-induced reduction in CAT expression was concurrent with the reduction in peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator (PGC)-1α and inactivation of PPARγ, both considered as positive regulators of CAT gene expression. Moreover, antioxidant N-acetylcysteine (NAC) demonstrated the capability to alleviate the downregulation of CAT gene expression both in vivo and in vitro. Additionally, NAC played a role in alleviating ATO-induced hepatotoxicity, potentially by mitigating the transcriptional downregulation of the CAT gene. Altogether, these results indicate that ATO exerts toxicity by inhibiting the antioxidant defense mechanism, which may be useful for forensic diagnosis of arsenic poisoning and clinical treatment of mitigating ATO-induced hepatotoxicity.

Asunto(s)

Acetilcisteína , Trióxido de Arsénico , Catalasa , Hígado , Óxidos , Trióxido de Arsénico/farmacología , Acetilcisteína/farmacología , Animales , Catalasa/metabolismo , Catalasa/genética , Ratas , Hígado/metabolismo , Hígado/efectos de los fármacos , Masculino , Arsenicales , Humanos , Expresión Génica/efectos de los fármacos , Antioxidantes/farmacología , Antioxidantes/metabolismo

RESUMEN

Duplex sequencing (DS) is an error-corrected next-generation sequencing (NGS) method that can overcome notorious high error rate from the process of NGS and detect ultralow-frequency mutations. In this study, we evaluated the mutagenicity of aristolochic acid, a known genotoxic carcinogen, and methapyrilene, a known nongenotoxic carcinogen using DS. Four male Fisher 344 rats were treated with aristolochic acid, methapyrilene, or the vehicle control for 6 weeks, liver tissues were collected one day after the treatment, and the DNA was isolated for analysis. The mutation frequency for the aristolochic acid-treated group was significantly increased over the vehicle control (44-fold), whereas no significant difference in the mutation frequency was observed between the methapyrilene-treated and the control groups. The primary type of mutation induced by aristolochic acid was A:T > T:A transversion, which occurred frequently at ApT sites, whereas the major type of mutation in the control and methapyrilene-treated groups was G:C > A:T transition, which occurred frequently at CpG sites. These findings are consistent with previously published data obtained with other in vivo mutation assays. Thus, our results suggest that the DS mutation assay is a promising technology for assessing mutagenicity of chemicals in vivo.

Asunto(s)

Ácidos Aristolóquicos , Metapirileno , Ratas , Animales , Masculino , Mutágenos/toxicidad , Ácidos Aristolóquicos/toxicidad , Carcinógenos/toxicidad

RESUMEN

N-nitrosamines, a very heterogeneous class of chemicals, may enter humans in small amounts through various sources and are produced endogenously, too. Some are known to be mutagenic carcinogens and have recently been detected as impurities in several marketed pharmaceuticals. Despite their known mutagenic properties, the suitability of the bacterial reverse mutation (Ames) assay and in particular the use of induced rat liver S9 to detect their mutagenic potential, is often discussed. Recently, it could be demonstrated that induced rat liver S9 is capable of metabolizing small alkyl nitrosamines to exert their mutagenic potential (Bringezu & Simon, 2022). In this project, the mutagenic potential of nitrosamines in vitro under different S9 conditions applying the preincubation protocol and OECD 471-compliant standard Ames test recommendations was investigated. These conditions included various amounts of S9 fraction from hamster and rat, uninduced or induced with Aroclor 1254 or Phenobarbital/beta-Naphthoflavone (PB/NF). The findings indicated that in addition to induced S9, uninduced hamster S9 also demonstrated effectiveness. Moreover, both rat and hamster S9 fractions exhibited suitable responses in terms of mutation frequencies. Increasing the S9 content did not increase the sensitivity of the Ames test. However, above 20% S9, reduced mutation frequency was observed in the higher concentration range suggesting cytotoxicity to the bacteria. Thus, limiting the S9 content to 10% provides reliable results and relates to a lower number of animals required for S9 production which is in concordance with the 3R principles (reduce, refine, replace) for animal testing. In addition, results obtained show that uninduced and induced hamster S9 are similarly effective, doubting the requirement of pretreating animals with enzyme inducers. Further investigations to compare mutagenicity data and rat and hamster S9 proteome analyses are ongoing.

RESUMEN

Oxidative stress is involved in a wide range of age-related diseases. A critical role has been proposed for mitochondrial oxidative stress in initiating or promoting these pathologies and the potential for mitochondria-targeted antioxidants to fight them, making their search and testing a very urgent task. In this study, the mitochondria-targeted antioxidants SkQ1, SkQ3 and MitoQ were examined as they affected isolated rat liver mitochondria and yeast cells, comparing SkQ3 with clinically tested SkQ1 and MitoQ. At low concentrations, all three substances stimulated the oxidation of respiratory substrates in state 4 respiration (no ADP addition); at higher concentrations, they inhibited the ADP-triggered state 3 respiration and the uncoupled state, depolarized the inner mitochondrial membrane, contributed to the opening of the mPTP (mitochondrial permeability transition pore), did not specifically affect ATP synthase, and had a pronounced antioxidant effect. SkQ3 was the most active antioxidant, not possessing, unlike SkQ1 or MitoQ, prooxidant activity with increasing concentrations. In yeast cells, all three substances reduced prooxidant-induced intracellular oxidative stress and cell death and prevented and reversed mitochondrial fragmentation, with SkQ3 being the most efficient. These data allow us to consider SkQ3 as a promising potential therapeutic agent to mitigate pathologies associated with oxidative stress.

Asunto(s)

Mitocondrias Hepáticas , Saccharomyces cerevisiae , Animales , Ratas , Antioxidantes/farmacología , Mitocondrias , Membranas Mitocondriales , Especies Reactivas de Oxígeno

RESUMEN

Our previous animal studies found that the preventive effects of lactoferrin (Lf) on alcoholic liver injury (ALI) are associated with nuclear factor E2-related factor 2 (Nrf2). To further explore the causality, experiments were performed using rat normal liver BRL-3A cells. Lf treatment reduced ethanol-induced death and apoptosis; meanwhile, Lf treatment alleviated excessive LDH release. These findings confirmed the protection of Lf against ethanol-induced injury in BRL-3A cells. Mechanistically, Lf treatment reversed the reduction in nuclear Nrf2 induced by ethanol without affecting the cytoplasmic Nrf2 level, which led to antioxidant enzyme activity restoration. However, the blocking of Nrf2 nuclear translocation by ML385 eliminated the protective effects of Lf. In a conclusion, Lf protects BRL-3A cells from ethanol-induced injury via promoting Nrf2 nuclear translocation.

Asunto(s)

Etanol , Lactoferrina , Ratas , Animales , Etanol/toxicidad , Etanol/metabolismo , Lactoferrina/farmacología , Lactoferrina/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Línea Celular , Hígado/metabolismo , Antioxidantes/farmacología , Estrés Oxidativo

RESUMEN

BACKGROUND: Mutagenicity, the ability of chemical agents to cause mutations and potentially lead to cancer, is a critical aspect of substance safety assessment for protecting human health and the environment. Metabolic enzymes activate multiple mutagens in living organisms, thus in vivo animal models provide highly important information for evaluating mutagenicity in human. Rats are considered suitable models as they share a similar metabolic pathway with humans for processing toxic chemical and exhibit higher responsiveness to chemical carcinogens than mice. To assess mutagenicity in rats, transgenic rodents (TGRs) are widely used for in vivo gene mutation assays. However, such assays are labor-intensive and could only detect transgene mutations inserted into the genome. Therefore, introducing a technology to directly detect in vivo mutagenicity in rats would be necessary. The next-generation sequencing (NGS) based error-corrected sequencing technique is a promising approach for such purposes. RESULTS: We investigated the applicability of paired-end and complementary consensus sequencing (PECC-Seq), an error-corrected sequencing technique, for detecting in vivo mutagenicity in the rat liver samples. PECC-Seq allows for the direct detection of ultra-rare somatic mutations in the genomic DNA without being constrained by the genomic locus, tissue, or organism. We tested PECC-Seq feasibility in rats treated with diethylnitrosamine (DEN), a mutagenic compound. Interestingly, the mutation and mutant frequencies between PECC-Seq and the TGR assay displayed a promising correlation. Our results also demonstrated that PECC-Seq could successfully detect the A:T > T:A mutation in rat liver samples, consistent with the TGR assay. Furthermore, we calculated the trinucleotide mutation frequency and proved that PECC-Seq accurately identified the DEN treatment-induced mutational signatures. CONCLUSIONS: Our study provides the first evidence of using PECC-Seq for in vivo mutagenicity detection in rat liver samples. This approach could provide a valuable alternative to conventional TGR assays as it is labor- and time-efficient and eliminates the need for transgenic rodents. Error-corrected sequencing techniques, such as PECC-Seq, represent promising approaches for enhancing mutagenicity assessment and advancing regulatory science.

RESUMEN

BACKGROUND: Liver cytochrome P450 (CYP) greatly contributes to the metabolism of endogenous substances and drugs. Recent studies have demonstrated that CYP expression in the liver is controlled by the central nervous system via hormonal pathways. In particular, the expression of hepatic CYPs is negatively regulated by the brain serotoninergic system. The present study aimed to investigate changes in the function of the main liver drug-metabolizing CYP enzymes as a result of serotonin depletion in the brain of aging rats, caused by knockout of brain tryptophan hydroxylase gene (TPH2-KO). METHODS: The hepatic CYP mRNA (qRT-PCR), protein level (Western blotting) and activity (HPLC), and serum hormone levels (ELISA) were measured in Dark Agouti wild-type (WT) male rats (mature 3.5-month-old and senescent 21-month-old) and in TPH2-KO senescent animals. RESULTS: The expression/activity of the studied CYPs decreased with age in the liver of wild-type rats. The deprivation of serotonin in the brain of aging males decreased the mRNA level of most of the studied CYPs (CYP1A/2A/2B/3A), and lowered the protein level of CYP2C11 and CYP3A. In contrast, the activities of CYP2C11, CYP3A and CYP2C6 were increased. The expression of cytochrome b5 decreased in aging rats, but increased in TPH2-deficient senescent animals. The serum concentration of growth hormone declined in the aged and further dropped down in TPH2-deficient senescent rats. CONCLUSIONS: Rat liver cytochrome P450 functions deteriorate with age, which may impair drug metabolism. The TPH2 knockout, which deprives brain serotonin, affects cytochrome P450 expression and activity differently in mature and senescent male rats.

Asunto(s)

Citocromo P-450 CYP3A , Serotonina , Ratas , Masculino , Animales , Serotonina/metabolismo , Citocromo P-450 CYP3A/metabolismo , Ratas Wistar , Sistema Enzimático del Citocromo P-450/metabolismo , Hígado , Encéfalo/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Envejecimiento , Microsomas Hepáticos/metabolismo

RESUMEN

The study aimed to investigate the hepatoprotective effects of purinergic receptor (P2X7R) antagonism by A438079 in liver damage. An experimental model of inflammation was performed by intraperitoneal (i.p.) lipopolysaccharide (LPS) administration in the rat. The groups were Control, A438079, dimethyl sulfoxide (DMSO), LPS, LPS + DMSO, and LPS + A438079. Following LPS (8 mg/kg) injection, A438079 (15 mg/kg) and DMSO (0.1 mL) were administrated (i.p) in the study groups. The blood and the liver tissues were removed for histological, biochemical, and western blot analyses. In the biochemical analysis, serum aspartate transaminase (AST) and alanine transaminase (ALT) concentrations, the tissue glutathione (GSH) level, and superoxide dismutase (SOD) activity dramatically decreased and malondialdehyde (MDA) level increased in the LPS and LPS + DMSO groups compared to the LPS + A438079 group. In the histological analysis, severe sinusoidal dilatation, necrotic hepatocytes, and inflammatory cell infiltration were observed in the LPS and LPS + DMSO groups while these effects were lessened in the LPS + A438079 group. The relative protein expression levels of P2X7R, Nf-kB-p65, IL-6, and Caspase-3 were significantly higher in the LPS and the LPS + DMSO groups than in the LPS + A438079 group. On the other hand, these protein expressions were considerably lower in the Control, A438079, and DMSO groups compared to the LPS + A438079 group. In addition, Bcl-2 protein expression was significantly lower in the LPS and the LPS + DMSO groups and higher in the LPS + A438079 group compared to the other groups. The protective effect of A438079 against LPS-induced hepatic inflammation may be related to P2X7R antagonism, inflammatory mediators, and apoptotic cell death.

Asunto(s)

Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Lipopolisacáridos , Ratas , Animales , Lipopolisacáridos/toxicidad , Receptores Purinérgicos P2X7/metabolismo , Dimetilsulfóxido/metabolismo , Dimetilsulfóxido/farmacología , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/patología , Hígado/metabolismo , Inflamación/metabolismo , Alanina Transaminasa

RESUMEN

BACKGROUND: Understanding the impact of altered hepatic uptake and/or efflux on the hepatobiliary disposition of the imaging agents [99mTc]Mebrofenin (MEB) and [153Gd]Gadobenate dimeglumine (BOPTA) is important for proper estimation of liver function. METHODS: A multi-compartmental pharmacokinetic (PK) model describing MEB and BOPTA disposition in isolated perfused rat livers (IPRLs) was developed. The PK model was simultaneously fit to MEB and BOPTA concentration-time data in the extracellular space, hepatocytes, bile canaliculi, and sinusoidal efflux in livers from healthy rats, and to BOPTA concentration-time data in rats pretreated with monocrotaline (MCT). RESULTS: The model adequately described MEB and BOPTA disposition in each compartment. The hepatocyte uptake clearance was much higher for MEB (55.3 mL/min) than BOPTA (6.67 mL/min), whereas the sinusoidal efflux clearance for MEB (0.000831 mL/min) was lower than BOPTA (0.0127 mL/min). The clearance from hepatocytes to bile (CLbc) for MEB (0.658 mL/min) was similar to BOPTA (0.642 mL/min) in healthy rat livers. The BOPTA CLbc was reduced in livers from MCT-pretreated rats (0.496 mL/min), while the sinusoidal efflux clearance was increased (0.0644 mL/min). CONCLUSION: A PK model developed to characterize MEB and BOPTA disposition in IPRLs was used to quantify changes in the hepatobiliary disposition of BOPTA caused by MCT pretreatment of rats to induce liver toxicity. This PK model could be applied to simulate changes in the hepatobiliary disposition of these imaging agents in rats in response to altered hepatocyte uptake or efflux associated with disease, toxicity, or drug-drug interactions.

Asunto(s)

Hígado , Compuestos Organometálicos , Ratas , Animales , Hígado/diagnóstico por imagen , Hígado/metabolismo , Hepatocitos , Compuestos Organometálicos/farmacocinética , Bilis , Transporte Biológico

RESUMEN

Increasing evidence indicated that liquid crystal monomers (LCMs) in liquid crystal displays can be released into the environment, and ubiquitously detected in environmental matrices and even human bodies. Yet databases regarding its uptake and distribution in mammals are lacking. In this study, four LCMs (namely 3dFB, 2OdF3B, 2teFT, and 6OCB) with various physiochemical properties and structures were selected as the target compounds. The LCMs were in vivo and in vitro exposed to mice and rat liver microsomes (RLM). LCMs were found in all mouse tissues, including brain. Pharmacokinetics parameters, Cmax-tissue/Cmax-blood, ranged from 27.5 to 214, indicating the preferential deposition of LCMs to tissues rather than blood. The LCMs distributed preferentially to lipophilic tissues, and relative mass contribution of LCMs from liver and adipose was 43-98 %. The physicochemical properties (i.e., Kow, molecular weight, and functional groups) had pronounced effect on distribution and accumulation of LCMs. The 2teFT with the highest Kow and molecular weight showed the relatively higher accumulation potential and half elimination time in all the tissues. The 6OCB containing cyano-group was more accumulative than the fluorinated 3dFB with the comparable Kow. In RLM assays, 2teFT and 6OCB were resistant to metabolic degradation. While 3dFB and 2OdF3B underwent rapid degradation with 93.7 % and 72.4 % being metabolized at 360 min. Findings in this study bear significant implications for the biomonitoring and overall risk evaluation of LCMs.

Asunto(s)

Cristales Líquidos , Ratones , Ratas , Humanos , Animales , Encéfalo/metabolismo , Microsomas Hepáticos , Hígado , Mamíferos

RESUMEN

Hyperlipidemia is a medical condition characterized by elevated levels of blood lipids, especially triglycerides (TG). However, it remains unclear whether TG levels remain consistently elevated throughout the entire developmental stage of the high-lipid state. In our animal experiment, we found that TG levels were significantly higher in the early stage of the high-lipid model but significantly decreased at the 14th week of the late stage, reaching levels similar to those of the control group. This suggests that TG levels in the high-lipid model are not always higher than those of the control group. To determine the reason for this observation, we used in situ mass spectrometry imaging (MSI) to detect the distribution of metabolites in the liver of rats. The metabolite distribution of the control rats at different stages was significantly different from that of the model rats, and the high-lipid model differed significantly from the control rats. We identified nine functional metabolites that showed differences throughout the period, namely, PA(20:3-OH/i-21:0), PA(20:4-OH/22:6), PG(20:5-OH/i-16:0), PG(22:6-2OH/i-13:0), PG(O-18:0/20:4), PGP(18:3-OH/i-12:0), PGP(PGJ2/i-15:0), SM(d18:0/18:1-2OH), and TG(14:0/14:0/16:0), among which TG was most significantly correlated with hyperlipidemia and high lipid. This study is unique in that it used MSI to reveal the changes in metabolites in situ, showing the distribution of different metabolites or the same metabolite in liver tissue. The findings highlight the importance of considering the animal's age when using TG as a biomarker for hyperlipidemia. Additionally, the MSI images of the liver in the high-lipid model clearly indicated the distribution and differences of more significant metabolites, providing valuable data for further research into new biomarkers and mechanisms of hyperlipidemia. This new pathway of in situ, visualized, and data-rich metabolomics research provides a more comprehensive understanding of the characteristics of high lipid and its implications for disease prevention and treatment.

RESUMEN

Heavy metals are ubiquitous environmental pollutants that are extremely dangerous for public health, but the molecular mechanisms of their cytotoxic action are still not fully understood. In the present work, the possible contribution of the mitochondrial ATP-sensitive potassium channel (mK(ATP)), which is usually considered protective for the cell, to hepatotoxicity caused by heavy metals was investigated using polarography and swelling techniques as well as flow cytometry. Using isolated liver mitochondria from adult male Wistar rats and various potassium media containing or not containing penetrating anions (KNO3, KSCN, KAcet, KCl), we studied the effect of mK(ATP) modulators, namely its blockers (5-hydroxydecanoate, glibenclamide, ATP, ADP) and activators (diazoxide, malonate), on respiration and/or membrane permeability in the presence of hepatotoxins such as Cd2+, Hg2+, and Cu2+. It has been shown for the first time that, contrary to Hg2+ and depending on media used, the mK(ATP) modulators affect Cd2+- and/or Cu2+-induced alterations in mitochondrial swelling and respiration rates, although differently, nevertheless, in the ways compatible with mK(ATP) participation in both these cases. On rat AS-30D ascites hepatoma cells, it was found that, unlike Cd2+, an increase in the production of reactive oxygen species was observed with the simultaneous use of Cu2+ and diazoxide; in addition, there was no protective effect of diazoxide against cell death, which also occurred in the presence of Cu2+. In conclusion, the relationships (functional, structural and/or regulatory) between mK(ATP), components of the mitochondrial electron transport chain (CI, CII-CIII and/or ATP synthase, CV) and mitochondrial permeability transition pores were discussed, as well as the role of these molecular structures in the mechanisms of the cytotoxic action of heavy metals.

Asunto(s)

Carcinoma Hepatocelular , Neoplasias Hepáticas , Mercurio , Metales Pesados , Ratas , Masculino , Animales , Mitocondrias Hepáticas , Canales KATP/metabolismo , Canales KATP/farmacología , Diazóxido/metabolismo , Diazóxido/farmacología , Cadmio/toxicidad , Ascitis/metabolismo , Carcinoma Hepatocelular/metabolismo , Ratas Wistar , Metales Pesados/metabolismo , Mercurio/metabolismo , Neoplasias Hepáticas/metabolismo , Adenosina Trifosfato/metabolismo

RESUMEN

Environmental pollutants, such as quinoline (QN) and 4-methylquinoline (4-MeQ), may be genotoxic and carcinogenic. Earlier studies, including in vitro genotoxicity tests, indicated that 4-MeQ is more mutagenic than QN. However, we hypothesized that the methyl group of 4-MeQ favors detoxication over bioactivation, and this factor may be overlooked in in vitro tests that do not incorporate supplementation with cofactors for enzymes that catalyze conjugation reactions. We used human induced hepatocyte cells (hiHeps), which express such enzymes, and compared the genotoxicity of 4-MeQ and QN. We also carried out an in vivo micronucleus (MN) test in rat liver, since 4-MeQ is not genotoxic in rodent bone marrow. In the Ames test and the Tk gene mutation assay, with rat S9 activation, 4-MeQ was more mutagenic than QN. However, QN induced significantly higher MN frequencies in hiHeps and rat liver than did 4-MeQ. Furthermore, QN upregulated genotoxicity marker genes much more than did 4-MeQ. We also investigated the roles of two important detoxication enzymes, UDP-glucuronosyltransferases (UGTs) and cytosolic sulfotransferases (SULTs). When hiHeps were preincubated with hesperetin (UGT inhibitor) and 2,6-dichloro-4-nitrophenol (SULT inhibitor), MN frequencies were elevated approximately 1.5-fold for 4-MeQ, whereas no significant effects were seen for QN. This study shows that QN is more genotoxic than 4-MeQ, when the roles of SULTs and UGTs in detoxication are considered and our results may improve understanding the structure-activity relationships of quinoline derivatives.

Asunto(s)

Mutágenos , Quinolinas , Animales , Humanos , Ratas , Núcleo Celular , Glucuronosiltransferasa , Hígado , Quinolinas/toxicidad