Old age was associated with higher serum levels of acetaminophen glucuronide, whereas serum acetaminophen sulfates was not significantly higher in old age rats compared with young rats Figure 1A. These findings are consistent with both patient 38 and rodent studies 35 and suggest either increased conjugation activity or accumulation due to impaired renal clearance.
Phase II metabolism is regulated by Nrf2, which did not vary with age or treatment in our study, and several studies have reported no change or a decline in conjugation activity with aging in humans 11 and rat livers We observed elevated serum creatinine a marker of renal function following acetaminophen treatment in old rats. Many studies have reported that acetaminophen can cause renal lesions and this risk is increased with age 35 , 36 , Together with existing literature, our findings indicate that it is unlikely that Phase II metabolism is increased with aging, instead the elevation of serum conjugation metabolites is likely to be the result of impaired renal clearance from acetaminophen-induced nephrotoxicity.
In this study, a variety of methods were employed to investigate the major recognized metabolic pathways of acetaminophen. The animal model selected is a high-quality aging model free from age-associated obesity as discussed by Rikans in Importantly, the observed lack of increase in susceptibility to acetaminophen hepatotoxicity in old age 39 and pharmacokinetic changes are consistent with human patient results 38 , supporting the clinical relevance of our model.
However, recently, rats have been proposed to be slightly more resistant to acetaminophen-induced hepatotoxicity than humans 6 , and future research may be necessary to confirm these findings in lean mice, which have similar susceptibility to humans.
Future research may also investigate other aging and antiaging models such as calorie-restricted and long-lived Ames dwarf mice and the effect of acetaminophen to better understand the mechanisms of acetaminophen-induced hepatotoxicity 50 , The time of drug exposure is an important parameter for hepatotoxicity studies. We selected an incubation time of 4 hours as the half-life of acetaminophen is approximately 3 hours 52 , which enabled us to observe changes in the concentrations of acetaminophen and its metabolites.
We also were aware that glutathione levels deplete prior to this time point 6 and hence we expected hepatotoxicity and any protective responses to commence within this period. Our animals were also fasted to normalize their nutritional stores and potentiate hepatotoxicity In conclusion, old age is not associated with increased risk of acetaminophen-induced hepatotoxicity in male Fischer rats despite higher serum levels of acetaminophen and its glucuronide metabolite.
Detailed investigation of the potential mechanisms found that old age is associated with toxokinetic changes that favor reduced formation and enhanced metabolism of NAPQI, with no observed changes in Nrf2 mediated resistance with age or treatment. Although old age is associated with a reduced risk of acetaminophen liver toxicity, the changes in acetaminophen pharmacokinetics result in increased serum acetaminophen, which may increase the exposure of the kidneys and result in acetaminophen-induced nephrotoxicity.
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J Clin Pharm Ther. Age-related pseudocapillarization of the liver sinusoidal endothelium impairs the hepatic clearance of acetaminophen in rats. Role of CYP2E1 in the hepatotoxicity of acetaminophen. J Biol Chem. Cytochrome P 2E1 expression induces hepatocyte resistance to cell death from oxidative stress. Antioxid Redox Signal. Protective effect of ethanol against acetaminophen-induced hepatotoxicity in mice: role of NADH:quinone reductase. Inhibitors of NQO1: Identification of compounds more potent than dicoumarol without associated off-target effects.
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Hine CM Mitchell J. NRF2 and the phase II response in acute stress resistance induced by dietary restriction. J Clin Expt Pathol. Nioi P Hayes JD. Contribution of NAD P H:quinone oxidoreductase 1 to protection against carcinogenesis, and regulation of its gene by the Nrf2 basic-region leucine zipper and the arylhydrocarbon receptor basic helix-loop-helix transcription factors.
Mutat Res. Acetaminophen and p-aminophenol nephrotoxicity in aging male Sprague-Dawley and Fischer rats. Long-lived ames dwarf mice are resistant to chemical stressors.
Stress resistance and aging: influence of genes and nutrition. Mech Ageing Dev. Increased resistance of diabetic rats to acetaminophen-induced hepatotoxicity. Journal of Pharmacology and Experimental Therapeutics. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search.
Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Experimental Procedures. John Mach , John Mach. Oxford Academic. Aniko Huizer-Pajkos. Victoria C Cogger. Catriona McKenzie. David G Le Couteur. Brett E Jones. Rafael de Cabo. Sarah N Hilmer. Email: sarah. Select Format Select format. Permissions Icon Permissions. Abstract We investigated the effect of aging on hepatic pharmacokinetics and the degree of hepatotoxicity following a toxic dose of acetaminophen.
Acetaminophen , Toxicity , Aging , Pharmacokinetics. The ligation product was transferred into E. The plasmid was transferred into E. At an OD of about 0. The supernatant was filtered through a 0. Cell free lysate was loaded onto the column, which was then washed with 15 column volumes of 20 mM Tris buffer pH 8 with mM NaCl and 6 mM imidazole.
Extinction coefficients for K 3 Fe CN 6 were 0. Fitting of enzyme kinetics according to appropriate models was performed with Excel Solver using a Levenberg-Marquardt fit for a Michealis-Menten kinetic model for substrate inhibition.
This model is equivalent to a model of uncompetitive inhibition, in which substrate and inhibitor concentrations are equated Equation 1. Substrate concentration [ S ] is also used for inhibitor concentration [ I ]. In enzyme assays, about 0. For assays with other electron acceptors, 1 ml assays were performed in 1. Assays with 0. Enzyme assays with cell extracts of E. To these assays, 0. To determine the position of the double bond, cell extract of E.
Louis, MO, United States. A gradient method starting with 0. Cholate concentration was determined from base peak chromatogram in negative mode MS measurements and standard curves.
Concentrations of steroid degradation intermediates were determined as peak areas in arbitrary units. For identification of analytes, molecular masses, and UV absorption spectra as well as retention times were consulted.
Prediction of protein domains was performed with Interpro Mitchell et al. Alignments for phylogenetic trees were calculated in MegaX Kumar et al. Phylogenetic trees were calculated in MegaX using maximum parsimony method and bootstrap validation with 50 repetitions. The presence of an iron-sulfur cluster was predicted with Interpro, comparisons and with metalpredator Valasatava et al.
During proteome analysis of Sphingobium sp. For further elucidation of Nov2c function, its gene was expressed from a plasmid in E. In enzyme assays with 3-ketocholate 5 in Figure 1 as substrate, K 3 Fe CN 6 as electron acceptor and cell extract of E. For determining the position of the double bond, cell extract of E. Hsh2 is a 7-hydroxysteroid dehydratase from Sphingobium sp. Upon addition of this cell extract, HOCDA 11 was formed as indicated by its characteristic absorption at nm and molecular mass of Da for the deprotonated acid Holert et al.
Figure 3. Samples were analyzed by high-performance liquid chromatography coupled to mass spectrometry HPLC-MS and steroid compounds were identified based on retention time, absorbance spectrum, and mass.
Masses are indicated for the respective deprotonated acids. Bioinformatic analyses predicted a relatively large protein of about 75 kDa belonging to the family of old yellow enzymes. These analyses also predicted two flavin cofactors as well as an [4Fe-4S] cluster involving cysteines C, C, C, and C Consistent with this, a protein with a mass of slightly more than 70 kDa Supplementary Figure S1 , absorbance maxima characteristic for flavin cofactors at about and nm Figure 4A and corresponding yellow color Figure 4B was purified.
Figure 4. B Yellow-colored solution of purified 2. Figure 5. Enzyme assays were analyzed by HPLC-MS and steroid compounds were identified by retention time, absorbance spectrum, and mass. Higher concentrations of the organic substrate led to strongly decreasing rates, indicating substrate inhibition Figure 6A.
Indeed, using a Michealis-Menten kinetic model for substrate inhibition, which is equivalent to a model of uncompetitive inhibition, in which substrate and inhibitor concentrations are equated, the data could be described accurately.
The Levenberg-Marquardt fit led to a v max of about A possible reason for the inhibitory effect of 3-ketocholate could be its detergent character that may affect enzyme functionality. We therefore undertook tests to determine the general susceptibility of the enzyme for detergent-induced unspecific inhibition. At a 3-ketocholate concentration of 0. Figure 6. The control contained no electron acceptor besides oxygen from air that is found in all assays.
However, no increase of activity with increasing substrate concentration could be observed, possibly because substrate saturation is already achieved at concentrations below detection limit of the spectrophotometric assay i. However, enzyme assays were restricted by low substrate solubility in water and ambiguous concentration determination because of limited purity of the commercial substrate.
Consistently, enzyme activity linearly increased with pH from 5. An unmarked deletion mutant of the corresponding gene, Sphingobium sp. This prolonged lag-phase was not observed when cells were grown with glucose data not shown. Both strains had similar growth rates and reached a similar final optical OD Cholate degradation by the deletion mutant and the wild type was very similar Figure 7B , but regarding the accumulation of degradation intermediates, more pronounced differences were observed.
Probably, burden from bearing of the plasmid interfered with the positive effect of the complementation. Figure 7. Growth of Sphingobium sp. To analyze cholate degradation and intermediate accumulation in more detail, suspensions of resting glucose-grown cells were supplied with cholate Figure 8. For investigating whether the prolonged lag-phase was due to a delayed upregulation of compensatory genes or lower activity of alternative enzymes, suspensions of glucose-grown cells were incubated with cholate and chloramphenicol Figure 9.
Here, cholate degradation by both strains was much slower with residual cholate detected even after 27 h. Figure 8. Degradation of cholate 1 in Figure 2 by suspensions of glucose-grown cells of Sphingobium sp. Figure 9. Chloramphenicol was added after preparation of cell suspensions prior to substrate addition.
Growth experiments with different cholate concentrations revealed that Sphingobium sp. With both, 2 and 3 mM cholate, Sphingobium sp. To further investigate cholate sensitivity of Sphingobium sp. However, no significant differences between Sphingobium sp. Whereas 1 mM cholate had no effect on both strains after 15 and 90 min, CFUs in cell suspensions of both strains incubated with 10 mM cholate decreased by a factor of 10 and after 15 and 90 min, respectively.
When incubated with 50 mM cholate, CFUs in both cell suspensions decreased by a factor of 10 6 after 15 min, and no CFUs could be detected after 90 min. In the genome of Sphingobium sp. However, neither homolog was detected during the proteome studies unpublished results. In each P. The respective genes coding for CasH from R.
Table 3. For testing the function of these homologs, selected genes were expressed in E. CasH from R. In contrast, no activity could be detected in enzyme assays with Nov2c and Nov2c, the homologs from Sphingobium sp. Accordingly, a respective unmarked deletion mutant of P.
Figure UV-chromatograms of enzyme assays with 3-ketocholate 5 in Figure 2 as substrate, K 3 Fe CN 6 as electron acceptor and cell extracts of E.
Chromatograms are depicted with an offset in both retention time and intensity for easier distinction. Within this cluster, homologs from several bile-salt degrading proteobacteria such as Sphingomonadaceae , P.
However, in the given selection of proteins, steroid dehydrogenases did not seem to be clustered separately from enzymes with other functions. The tree was constructed based on ClustalW alignment and maximum parsimony method. Branch colors indicate bootstrap values as indicated. Proteins were chosen from BLAST searches among well characterized proteins with verified function or structure or from known bile-salt degrading bacteria.
With respect to the toxicity of bile salts as growth substrates, low K m and high turnover of the substrate are obvious advantages for a fast alleviation of the toxic properties by transformation of the bile salts.
The necessity for a fast detoxification mechanism in Sphingobium sp. The fact that glucose-grown cells of Sphingobium sp.
Additionally, it also showed that the isoenzymes need to be upregulated, which is further supported by the strongly delayed transformation of cholate into HOCDA 11 in the presence of the translation-blocking chloramphenicol. While substrate inhibition is apparently a very widespread phenomenon, its physiological functions are mainly unknown but appear to be very diverse Reed et al.
These functions also comprise the homoeostasis of metabolic pathways. Depletion of free CoA has been hypothesized to be the reason for toxicity of cholesterol in Mycobacterium tuberculosis mutants lacking genes involved in degradation of HIPs 10 in Figure 2 ; Crowe et al. In Sphingobium sp. Based on these considerations, substrate inhibition by 3-ketocholate 5 in Figure 2 can be interpreted as a circuit breaker of bile-salt metabolism.
Curbing or even shutting down bile-salt catabolism by substrate inhibition would create high levels of intracellular bile-salts and would also shut down growth. In agreement with that, Sphingobium sp. Of course, this shutdown could also at least partly be due to toxic effects exerted by cholate. Therefore, the cells must be able to quickly reduce bile-salt levels by pumping them out of the cell.
To date, no specific efflux pumps in bile-salt degrading bacteria have been identified. However, as the transient accumulation of intermediates of bile-salt degradation is a common phenomenon in the respective bacteria it appears justified to postulate such efflux pumps to be very important for enabling growth with these toxic substrates Swain et al. In addition, further-downstream metabolites of cholate degradation might also have toxic effects on the cells, and substrate inhibition would therefore prevent the potential accumulation of these metabolites.
This conclusion is in agreement with earlier enzymatic studies with cell extracts of Sphingobium sp. In contrast, BaiCD from C. However, the additional two homologs in Sphingobium sp. A possible explanation could be that these enzymes process CoA-esters of 3-ketobile salts which could not be provided in these assays. However, the aforementioned substrate specificity of the CoA-ligase contradicts this theory.
It is unlikely that these genes could not functionally be expressed in E. These results indicate that yet unknown alternate enzymes catalyzing this reaction must exist in some bile-salt degrading bacteria that differ significantly in their sequence and probably belong to a different protein family.
Of all tested homologs, only CasH from a steroid degradation cluster in R. CasH is encoded in a cluster of cholate degradation genes, in proximity to many cas genes coding for steroid carboxylic C 5 side chain degradation enzymes Mohn et al.
Nevertheless, these include the major intestinal cholesterol transformation product coprostanol as well as many steroidal toxins. Additionally, bile-salt degradation is also observed with other type strains from this family unpublished results. FF conceptualized the study together with BP, planned and performed most of the experiments, wrote the first draft of the manuscript, and also supervised GM.
GM constructed the deletion mutant and performed the corresponding experiments. LW performed proteome studies. SD performed protein purification, gel filtration, and helped with evaluations of enzyme assays. BP was involved in conceptualization of the study, planning of the experiments, and writing of the manuscript.
All authors contributed to the manuscript revision and approved its submission. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Karin Niermann and Kirsten Heuer for excellent experimental support. Altschul, S. Basic local alignment search tool.
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