Biotransformation Kinetics Research Articles

Microbial transformations of S-naproxen by Aspergillus niger ATCC 9142.

Aspergillus niger ATCC 9142 was used to catalyze the biotransformation of S(-)-naproxen (1) to three major metabolites that were isolated by solvent extraction, purified chromatographically, and characterized by mass spectrometry and NMR spectroscopy. Metabolites were identified as O-desmethylnaproxen (2), 7-hydroxynaproxen (3) and 7-hydroxy-O-desmethyinaproxen (4). The kinetics of naproxen biotransformation to 2 and 4 was established over an 84 h period to show that naproxen was completely metabolized at 36 h, the major metabolite was O-desmethylnaproxen at 24 h, and the 7-hydroxy-O-desmethylnaproxen that was formed after 24 h.

Biogeochemistry of Aromatic Surfactants in Microtidal Estuaries

This paper summarizes several studies on input, distribution, and fate of two different types of aromatic surfactants, the anionic linear alkylbenzene sulphonates (LAS) and nonionic alkylphenol polyethoxylates (APnEO), in a karstic estuary characterized by sharp salinity gradients. A combination of reversed-phase and normal-phase HPLC analysis followed by spectrofluorimetric detection allowed detailed characterization of homologue and oligomer compositions of aromatic surfactants in wastewater and estuarine waters. The distribution patterns of homologues and/or oligomers in the wastewater plume reveal a significant alteration of the original composition of both surfactants by biotransformation and physico-chemical partitioning. The vertical distribution of surfactant residues in the estuarine water column is characterized by pronounced maxima at the air–brackish water and brackish water–sea water interface. The brackish water–sea water interface is a very efficient barrier, which prevents spreading of surfactants into the deeper layers. In addition, the interface seems to be a site of high biological activity, including biotransformation of surfactant residues. As a consequence, this layer can contain enhanced levels of lipophilic metabolites of APnEO. Biotransformation is the main mechanism for the removal of both surfactant classes from the water column. However, our studies suggest that a significant percentage of surfactant residues discharged into the stratified estuaries reaches the coastal sea due to the incomplete removal in the estuary. This situation is a consequence of a rather fast flushing of the brackish layer and a very slow biotransformation kinetics in the saline layer, especially in the winter period.

Aqueous Phase Biodegradation Kinetics of 10 PAH Compounds

Biodegradation kinetics were individually studied for 10 polycyclic aromatic hydrocarbons (PAHs): naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, 2-ethylnaphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Experiments consisted of aerobic, continuously mixed, batch aqueous systems inoculated with a PAH-degrading consortium. The design of the experiments and the mathematical interpretation of the data isolated the kinetics of biotransformation. Other physical-chemical processes that may affect bioavailability were eliminated or independently measured. Model parameters were estimated using the maximum likelihood method for a bivariate data set of substrate and biomass concentration over time. For four of the PAHs, naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, and 2-ethyl naphthalene, there was sufficient growth to estimate biomass yield coefficients. For two of the PAHs, naphthalene and 1-methylnaphthalene, Monod parameters qmax and Ks were estimated. For the remaining compounds, only the first-order rate coefficients were estimated. The consortium was able to degrade all the PAHs except acenaphthene. The observed biodegradation rate coefficients did not correlate with molecular weight or any other chemical property. The biomass-normalized first-order biodegradation rate coefficients spanned a little less than two orders of magnitude, from 0.0255 to 1.11 h-1 (mg protein/L)-1. This is much less variation than is typically observed for PAHs in soils and sediments. This suggests that in field samples differences in biodegradation rates are not governed by differences in the kinetics of biotransformation. Rather, the differences in biodegradation rates in the field are primarily governed by the physical-chemical processes that control bioavailability, which differ significantly for different PAHs.

Characterization and application of xylene monooxygenase for multistep biocatalysis.

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275:10085-10092, 2000). Using E. coli expressing xylMA, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of E. coli was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based two-liquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.

Stereoselective metabolism of pentoxifylline in vitro and in vivo in humans.

Pentoxifylline increases erythrocyte flexibility, reduces blood viscosity, and inhibits platelet aggregation and is thus used in the treatment of peripheral vascular disease. It is transformed into at least seven phase I metabolites, of which two, M1 and M5, are active. The reduction of the keto group of pentoxifylline to a secondary alcohol in M1 takes place chiefly in erythrocytes, is rapidly reversible, and creates a chiral center. The aims of this study were: to develop HPLC methods to separate the enantiomers of M1, to investigate the kinetics of the reversible biotransformation of pentoxifylline to (R)- and (S)-M1 in hemolysed erythrocyte suspension, and to quantify the formation of the enantiomers of M1 (as well as M4 and M5) after intravenous and oral administration of pentoxifylline to human volunteers. (R)- and (S)-M1 could be separated preparatively on a cellobiohydrolase column, while determination in blood or plasma was by HPLC after chiral derivatization with diacetyl-L-tartaric acid anhydride. The metabolism of pentoxifylline to (R)-M1 in suspensions of hemolysed erythrocytes followed simple Michaelis-Menten kinetics (K(m) = 11 mM), while that to (S)-M1 was best described by a two-enzyme model (K(m) = 1.1 and 132 mM). Studies with inhibitors indicated that the enzymes were of the carbonyl reductase type. At a therapeutic blood concentration of pentoxifylline, the calculated rate of formation of (S)-M1 is 15 times higher than that of the (R)-enantiomer. Back-conversion of M1 to pentoxifylline was 3-4 times faster for the (S)- than for the (R)-enantiomer. In vivo, the R:S plasma concentration ratio of M1 ranged from 0.010-0.025 after intravenous infusion of 300 or 600 mg of pentoxifylline, and from 0.019-0.037 after oral administration of 600 mg. The biotransformation of pentoxifylline to M1 was thus highly stereoselective in favor of the (S)-enantiomer both in vitro and in vivo.

In vitro metabolism and drug interaction potential of a new highly potent anti‐cytomegalovirus molecule, CMV423 (2‐chloro 3‐pyridine 3‐yl 5,6,7,8‐tetrahydroindolizine 1‐carboxamide)

To identify the enzymes involved in the metabolism of CMV423, a new anticytomegalovirus molecule, to evaluate its in vitro clearance and to investigate its potential involvement in drug/drug interactions that might occur in the clinic. The enzymes involved in and the kinetics of CMV423 biotransformation were determined using pools of human liver subcellular fractions and heterologously expressed human cytochromes P450 (CYP) and FMO. The effect of CMV423 on CYP probe activities as well as on indinavir and AZT metabolism was determined, and 26 drugs were tested for their potential to inhibit or activate CMV423 metabolism. CMV423 was oxidized by CYP and not by FMO or cytosolic enzymes. The Km values for 8-hydroxylation to rac-RPR 127025, an active metabolite, and subsequent ketone formation by human liver microsomes were 44 +/- 13 microM and 47 +/- 11 microM, respectively, with corresponding Vmax/Km ratios of 14 and 4 microl min(-1) nmol(-1) P450. Inhibition with selective CYP inhibitors indicated that CYP1A2 was the main isoform involved, with some participation from CYP3A. Expressed human CYP1A1, 1A2, 2C9, 3A4 and 2C8 catalysed rac-RPR 127025 formation with Km values of < 10 microM, 50 +/- 21 microM, 55 +/- 19 microM, circa 282 +/- 61 microM and circa 1450 microM, respectively. CYP1B1, 2A6, 2B6, 2C19, 2D6, 2E1 or 3A5 did not catalyse the reaction to any detectable extent. CYP1A1 and 3A4 also catalysed ketone formation from rac-RPR 127025. In human liver microsomes, CMV423 at 1 and 10 microM inhibited CYP1A2 activity up to 31% and 63%, respectively, CYP3A4 activity up to 40% (10 microM) and CYP2C9 activity by 35% (1 and 10 microM). No effect was observed on CYP2A6, 2D6 and 2E1 activities. CMV423 had no effect on indinavir and AZT metabolism. Amongst 26 drugs tested, none inhibited CMV423 metabolism in vitro at therapeutic concentrations. CMV423 is mainly metabolized by CYP1A2 and 3A4. Its metabolism should not be saturable at the targeted therapeutic concentrations range (Cmax < 1 microM). CMV423 will probably affect CYP1A2 and 1A1 activities in vivo to some extent, but no other drug-drug interactions are expected.

Biotransformation kinetics of Pseudomonas putida for cometabolism of phenol and 4-chlorophenol in the presence of sodium glutamate.

A kinetic model to describe the degradation of phenol and cometabolic transformation of 4-chlorophenol (4-cp) in the presence of sodium glutamate (SG) has been developed and validated experimentally. The integrated model accounts for cell growth, toxicity of 4-cp, cross-inhibitions among the three substrates, and the different roles of the specific growth substrate (phenol) and the conventional carbon source (SG) in the cometabolism of 4-cp. In this ternary substrate system, the overall phenol degradation and 4-cp transformation rates are greatly enhanced by the addition of SG since SG is able to attenuate the toxicity of 4-cp and therefore increase the cell growth rate. Model analysis indicates that the maximum specific degradation rate of phenol (0.819 mg (mg.h)(-1)) is lowered by SG by up to 46% whereas the specific transformation rate of 4-cp is not directly affected by the presence of SG. The competitive inhibition coefficient of 4-cp to phenol degradation (Ki,cp) and that of phenol to 4-cp transformation (Ki,ph) were determined to be 6.49 mg l(-1) and 0.193 mg l(-1), respectively, indicating that phenol imposes much larger competitive inhibition to 4-cp transformation than the converse. The model developed can simultaneously predict phenol degradation and 4-cp transformation, and is useful for dealing with cometabolism involving multiple substrates.

Tolerance to nitrogenous explosives and metabolism of TNT by cell suspensions of Datura innoxia

Cell suspension cultures of Datura innoxia were incubated in the presence of the nitro-substituted explosives 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5-triazine (RDX), and 1,3,5,7-tetranitro-1,3,5,7-tetraazocyclooctane (HMX). Cellular tolerance levels and TNT biotransformation kinetics were examined. Tolerance to TNT varied as cell suspensions aged. Concentrations of RDX or HMX in excess of reported solubility limits produced no observable changes in cell viability. GC/MS analysis of TNT-treated cell media and cell lysates revealed rapid removal of TNT. Within 12 h, less than 1% of the initial TNT remained in the growth medium. Aminodinitrotoluenes (ADNTs), known metabolites of TNT, accumulated transiently in cell lysates, and to a lesser extent in cell media. ADNT concentrations started to decrease after 3 h. After 12 h, less than 5% of the initial TNT could be detected as ADNT. Total ADNTs never exceeded 26% of initial TNT, suggesting that additional biotransformation steps also occurred. No other nitroaromatics were detected. A pseudo-first order rate constant for TNT clearance was calculated, k=0.40 h−1. D. innoxia cell suspension cultures demonstrated virtually complete clearance of TNT and of subsequent ADNT metabolites in less than 12 h. This rapid metabolism of nitroaromatics by the Datura cell suspension system indicates the utility of this system for further molecular and biochemical studies.

Effects of oxazepam and acetaminophen on cicletanine metabolism in rat hepatocytes and liver microsomes.

Cicletanine, a racemic furopyridine derivative synthesized as racemate, is used as an antihypertensive agent. Its two enantiomers are involved in the pharmacological effects of the drug. Cicletanine is metabolized by conjugation enzyme systems (phase II) into sulfoconjugated or glucuroconjugated enantiomers. As oxazepam and acetaminophen are widely prescribed, especially to elderly patients, these two drugs may be co-administered with cicletanine. The metabolic profile and the kinetics of biotransformation were studied by using rat hepatocytes and liver microsomes. Cicletanine was extensively metabolized by rat hepatocytes. More than 80% of the drug was biotransformed after a 3 h incubation. The formation of glucuroconjugated metabolites was characterized by the following kinetic parameters, i.e. Vmax = 2.05 +/- 0.21 nmol/min/mg protein and Km = 287 +/- 6.7 microM for (-)-cicletanine, and Vmax = 1.44 +/- 0.12 nmol/min/mg protein and K(m) = 171 +/- 4.1 microM for (+)-cicletanine. Oxazepam inhibited the glucuronidation of cicletanine in both rat hepatocytes and liver microsomes with a competitive-type inhibition, i.e. K(i) = 129 +/- 7.5 and 152 +/- 19.7 microM for (-)-cicletanine and (+)-cicletanine, respectively. The co-incubation of acetaminophen with cicletanine showed that only sulfoconjugation was inhibited in rat hepatocytes. Glucuronidation was not modified by acetaminophen. As natriuric activity is due to sulfoconjugated (+)-cicletanine, acetaminophen could potentially modulate in vivo the pharmacological effect of cicletanine. The data of the in vitro study reported here suggested an interaction between cicletanine and oxazepam or cicletanine and acetaminophen. However, the clinical impact of such a drug interaction needs further evaluation.

In vitro biotransformation of atrazine by rat liver microsomal cytochrome P450 enzymes

We studied atrazine (ATZ) metabolism in male and female rat liver microsomes in vitro, and the major metabolite was deisopropylatrazine (DeiPr-ATZ) with deethylatrazine (DeEt-ATZ) and 1-hydroxyisopropylatrazine (iPrOH-ATZ) as minor metabolites in both sexes. The enzyme kinetics of ATZ biotransformation were examined by means of Eadie–Hofstee analyses. Although no remarkable sex difference of Michaelis–Menten values for each pathway was observed, Cl ints ( V max/ K m) for DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ were slightly higher in female than in male rats. The formation of DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ from ATZ was substantially inhibited by SKF-525A, metyrapone, diallyl sulfide, 7-ethoxycoumarin, benzphetamine, nicotine, testosterone and lauric acid in both sexes. Cimetidine effectively inhibited the formation of all metabolites in male rats. On the other hand, the inhibition rates of the formation of DeiPr-ATZ and iPrOH-ATZ by cimetidine in female rats were lower than those in male rats, and DeEt-ATZ was hardly affected by the chemicals. In contrast with the results for cimetidine, the inhibition of ATZ biotransformation by bufuralol was more effective in female than in male rats. Anti-rat CYP2B1 and CYP2E1 antibodies effectively inhibited DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ formations in both sexes. Anti-rat CYP2C11 antibody also inhibited the three metabolites in both sexes, with the inhibition rates higher in male than in female rats, similar to cimetidine. In the case of anti-rat CYP2D1 antibody, the inhibitory effect on ATZ biotransformation in male rats was less than that in female rats. On the other hand, anti-rat CYP1A2, CYP3A2 and CYP4A1 antibodies did not affect the ATZ biotransformation in either sex. There was no significant correlation between the formation rate of ATZ metabolites and P450 isoform levels in either sex. These results may mean that CYP2B2, CYP2C11, CYP2D1 (only iPrOH-ATZ formation) and CYP2E1 in male rats, and CYP2B2, CYP2D1 and CYP2E1 in female rats are involved ATZ metabolism in liver, and that the substrate specificity of P450 isoforms for ATZ is broad.

Aerobic growth on nitroglycerin as the sole carbon, nitrogen, and energy source by a mixed bacterial culture.

Nitroglycerin (glycerol trinitrate [GTN]), an explosive and vasodilatory compound, was metabolized by mixed microbial cultures from aeration tank sludge previously exposed to GTN. Aerobic enrichment cultures removed GTN rapidly in the absence of a supplemental carbon source. Complete denitration of GTN, provided as the sole C and N source, was observed in aerobic batch cultures and proceeded stepwise via the dinitrate and mononitrate isomers, with successive steps occurring at lower rates. The denitration of all glycerol nitrate esters was found to be concomitant, and 1, 2-glycerol dinitrate (1,2-GDN) and 2-glycerol mononitrate (2-GMN) were the primary GDN and GMN isomers observed. Denitration of GTN resulted in release of primarily nitrite-N, indicating a reductive denitration mechanism. Biomass growth at the expense of GTN was verified by optical density and plate count measurements. The kinetics of GTN biotransformation were 10-fold faster than reported for complete GTN denitration under anaerobic conditions. A maximum specific growth rate of 0.048 +/- 0.005 h-1 (mean +/- standard deviation) was estimated for the mixed culture at 25 degreesC. Evidence of GTN toxicity was observed at GTN concentrations above 0. 3 mM. To our knowledge, this is the first report of complete denitration of GTN used as a primary growth substrate by a bacterial culture under aerobic conditions.

Extrapolation of in vitro enzyme induction data to humans in vivo

Enzyme induction generally increases the rate and extent of xenobiotic metabolism in vitro, but physiological constraints can dampen these effects in vivo. Biotransformation kinetics determined in hepatocytes in vitro can be extrapolated to whole animals based on the hepatocellularity of the liver, since the initial velocity of an enzyme-catalyzed reaction is directly proportional to the total enzyme present in the cell. The biotransformation kinetics of various xenobiotics determined with isolated hepatocytes in vitro have been shown to accurately predict pharmacokinetics in whole animals. Analysis of the kinetic data, using physiologically based pharmacokinetics, allows extrapolation of xenobiotic biotransformation across dose routes and species in a biologically realistic context. Several fold variations were observed in the bioactivation of the hepatotoxicant furan by isolated human hepatocytes, due to induction of cytochrome P450 2E1. Extrapolation of these data to humans in vivo showed that furan bioactivation was limited by hepatic blood flow delivery of the substrate. One important consequence of hepatic blood flow limitation is that the amount of metabolite formed in the liver is unaffected by increases in V max due to enzyme induction. Therefore, interindividual variations in cytochrome P450 2E1 among human populations would not affect the bioactivation of many rapidly metabolized hazardous chemical air pollutants. The hepatic blood flow limitation of biotransformation is also observed after oral bolus dosing of rapidly metabolized compounds. More slowly metabolized xenobiotics, such as therapeutic agents, are only partially limited by hepatic blood flow and other processes.

Modeling of the fate and effect of chlorinated phenols in anaerobic treatment processes

Studies were conducted to assess the biotransformation kinetics and inhibitory effects of mono-, di-, and trichlorophenol on acetogenic and methanogenic reactions using a standardized anaerobic protocol (Young and Tabak, 1993). Intrinsic kinetic parameters were estimated for each reaction using the Monod model. Reductive dechlorination of the toxicants was investigated using acclimated mixed cultures under methanogenic conditions. The studies indicated that inhibition of acetogenesis and methanogenesis was best described using exponential rather than linear inhibition terms. A second significant observation was that acclimated test cultures had a finite biodegradation capacity for each chemical that was related to the mass of microorganism present. A unique feature of the test program was that inhibition and biodegradation were modeled using the specific biomass responsible for acetogenesis and methanogenesis rather than the total anaerobic biomass.

Isocratic high-performance liquid chromatographic method for the separation of isradipine and its main metabolites. Application to in vitro metabolization by h3A4/OR cells.

A reversed-phase HPLC method was developed for the study of isradipine oxidation in vitro. The drug and its main metabolites were determined after extraction from culture media and from h3A4/OR cells having the cytochrome P450 isoenzyme involved in the xenobiotic metabolization. The HPLC assay was fully validated and was used to follow the biotransformation kinetics of isradipine.

Modeling of the fate and effect of chlorinated phenols in anaerobic treatment processes

Studies were conducted to assess the biotransformation kinetics and inhibitory effects of mono-, di-, and trichlorophenol on acetogenic and methanogenic reactions using a standardized anaerobic protocol (Young and Tabak, 1993). Intrinsic kinetic parameters were estimated for each reaction using the Monod model. Reductive dechlorination of the toxicants was investigated using acclimated mixed cultures under methanogenic conditions. The studies indicated that inhibition of acetogenesis and methanogenesis was best described using exponential rather than linear inhibition terms. A second significant observation was that acclimated test cultures had a finite biodegradation capacity for each chemical that was related to the mass of microorganism present. A unique feature of the test program was that inhibition and biodegradation were modeled using the specific biomass responsible for acetogenesis and methanogenesis rather than the total anaerobic biomass.

Prediction of Furan Pharmacokinetics from Hepatocyte Studies: Comparison of Bioactivation and Hepatic Dosimetry in Rats, Mice, and Humans

Furan is a volatile solvent and chemical intermediate that is hepatotoxic and hepatocarcinogenic in rats and mice but is not mutagenic or DNA-reactive. Furan hepatotoxicity requires cytochrome P450 2E1 bioactivation tocis-2-butene-1,4-dial. We have previously shown that furan biotransformation kinetics determined with freshly isolated rat hepatocytesin vitroaccurately predict furan pharmacokineticsin vivo[Kedderiset al.(1993)Toxicol. Appl. Pharmacol.123,274], suggesting that furan biotransformation kinetics determined with freshly isolated mouse or human hepatocytes can be used to develop species-specific pharmacokinetic models. Hepatocytes from male B6C3F1 mice or human accident victims (n= 3) were incubated with furan vapors to determine the kinetic parameters for furan bioactivation and compared to our previous data for rat hepatocytes. Isolated hepatocytes from all three species rapidly metabolized furan (Vmaxof 48 nmol/hr/106mouse hepatocytes, 19–44 nmol/hr/106human hepatocytes, and 18 nmol/hr/106rat hepatocytes) with high affinity (KMranging from 0.4 to 3.3 μM). The hepatocyte kinetic data and physiological parameters from the literature were used to develop dosimetry models for furan in mice and people. The hepatocyteVmaxvalues were extrapolated to whole animals assuming 128 × 106hepatocytes/g rodent liver and 137 × 106hepatocytes/g human liver. Simulations of inhalation exposure to 10 ppm furan for 4 hr indicated that the absorbed dose (mg/kg), and consequently the liver dose ofcis-2-butene-1,4-dial, was approximately 3- and 10-fold less in humans than in rats or mice, respectively. These results indicate that the target organ concentration, rather than the exposure concentration, is most appropriate for interspecies comparison of dose. The initial rates of furan oxidation in rat, mouse, and human liver were approximately 13-, 24-, and 37-fold greater than the respective rates of blood flow delivery of furan to the liver after 4-hr exposures to ≤300 ppm. One important consequence of blood flow limitation of furan bioactivation is that the amount of toxic metabolite formed in the liver will be unaffected by increases inVmaxdue to the induction of cytochrome P450 2E1. Therefore, the interindividual variations observed in cytochrome P450 2E1 activity among human populations would not be expected to have a significant effect on the extent of furan bioactivation in people. These considerations may be important for human cancer risk assessments of other rapidly metabolized rodent carcinogens.

Perifusion of co-cultured hepatocytes: optimization of studies on drug metabolism and cytotoxicity in vitro

The combination of co-cultivation of hepatocytes and epithelial cell lines with a newly developed perifusion system was used for in vitro studies on drug metabolism and cytotoxicity. This approach improved the viability and enhanced the induction of the biotransforming capacity of the hepatocytes. As demonstrated for the induction of 7-ethoxyresorufin O-deethylase activity by 3-methylcholanthrene or benzanthracene, co-cultured hepatocytes in the perifusion system responded more sensitively to these inducers than without perifusion, most likely owing to stable (steady-state) concentrations of the inducers under the former conditions and rapidly declining concentrations under the latter conditions. The perifusion approach rendered it possible to determine the kinetics of drug metabolism during single or sequential incubations. After induction with 3-methylcholanthrene and phenobarbital, phase I metabolism of lonazolac to the monohydroxylated product in perifused co-cultures closely (87%) approached the values reported for the in vivo production, whereas in stationary co-cultures only 52% could be reached. Likewise, cytotoxic effects could be detected more precisely in the perifused co-cultures. If cells were pretreated with 0.2 mmol/L galactosamine for 3 h, perifusion with increasing concentrations of menadione differentially killed epithelial RL-ET-14 cells and hepatocytes at low and high concentrations, respectively, while in stationary co-cultures no differential effect was observed and only the higher concentrations were cytotoxic for both cells. Prevention by incubation with S-adenosylmethionine of menadione cytotoxicity up to a menadione concentration of 250 micromol/L was seen only in the perifused co-cultures, whereas in stationary cultures only a slight shift of the cytotoxic concentration exerting 50% cell damage to higher values was noted. These results demonstrate the versatile application of perifused co-cultures for studies on drug metabolism including induction of cytochrome P450-dependent enzymes and steady-state kinetics of biotransformation, as well as cytotoxic and protective effects of different drugs.

Tacrolimus (FK 506) biotransformation in primary rat hepatocytes depends on extracellular matrix geometry.

Established in vitro models for studies of hepatic drug biotransformation include the use of primary hepatocytes. In normal liver the space of Disse provides the possibility of bilateral attachment to extracellular matrix for each hepatocyte. This configuration is disrupted by the cell isolation procedure of normal liver tissue, which delivers suspensions of round shaped cells. In standard culture configurations this unphysiologic cell shape terminates in a morphological dedifferentiation and inability to biotransform drugs. This study analyses the relevance of extracellular matrix geometry in hepatocyte monolayer configurations for expression and activity of cytochrome P450 3A. This enzyme is involved in the biotransformation of a large number of pharmaceuticals including the immunosuppressants tacrolimus and sirolimus. Morphological analysis of primary rat hepatocytes cultured with and without overlay of collagen type I was performed by transmission and scanning electron microscopy. Expression and activity of cytochrome P450 3A was studied by Western blot and the use of two model drugs specific for this enzyme. To this purpose the immunosuppressive drugs tacrolimus and sirolimus were used. Metabolites were analyzed by HPLC and HPLC/MS. Two sided attachment to extracellular matrix induces profound changes of the hepatocellular morphology in vitro resulting in the reconstitution of a polyhedric cell shape. This phenomenon is paralleled by an enhanced expression of cytochrome P450 3A and corresponding metabolic activity. As shown for tacrolimus biotransformation, the model may be useful to study complex metabolic patterns. In addition this model may facilitate studies of the kinetics of hepatocellular drug biotransformation in a setting with prolonged stability.

Biotransformation of nonylphenol polyethoxylate surfactants by estuarine mixed bacterial cultures

Biotransformation of nonylphenol polyethoxylates (NPnEOs), a major class of non-ionic surfactants, was studied under laboratory conditions by using a static die-away method. The experiments were performed with autochthonous bacterial cultures that originated from two essentially different estuarine compartments, i.e., from the brackish water and saline water layers of the highly stratified Krka River estuary, Croatia. Highly specific HPLC methods were applied to study biotransformation kinetics of individual NPnEO oligomers in the range of 1–16 EO units. Mixed bacterial cultures from the brackish water layer exhibited a significantly greater ability to transform NPnEO than that from the saline water layer. The results suggest, however, that this difference should not be interpreted as a salinity effect, but rather as a consequence of a better preadaptation of the brackish water bacteria to NPnEOs in their natural habitat. The biodegradation rates, calculated by presuming first-order kinetics, showed a strong temperature dependence. The estimated overall half-lives of NPnEOs under winter temperature conditions (13°C) ranged from 23–69 days, while the half-lives determined under summer temperature conditions (22.5°C) ranged from 2.5–35 days. Transformation kinetics of larger, more hydrophilic, oligomers of NPnEOs was faster than that of the lower ones. In fact, the elimination of higher NPnEOs was followed by a significant formation of the short-chain NPnEOs (nEO = 1–4). The main lipophilic intermediate formed during the experiments was nonylphenol diethoxylate (NP2EO), whose concentration in the medium increased by a factor of 10–20. Its relatively fast accumulation was followed by a rather slow degradation. Consequently, residual concentrations of NP2EO were detected in the medium even after 30 days.

Kinetics of alachlor and atrazine biotransformation under various electron acceptor conditions

AbstractSecond‐order rate constants were developed for the biotransformation of the herbicides atrazine and alachlor under aerobic, nitrate‐reducing, sulfate‐reducing, and methanogenic conditions. Batch‐reactor techniques were used, with seed cultures taken from acclimated biofilm columns. The reactors were fed acetate as a primary substrate. Pesticide biotransformation appeared to depend upon the continued presence of the primary substrate, indicating co‐metabolic transformations. All four electron acceptor conditions tested resulted in some biotransformation of both pesticides. Rate constants for alachlor ranged from 6.25 × 10−5 to 1.60 × 10−4 L/mg volatile suspended solids (VSS)·h, with the highest rate constant occurring under sulfate‐reducing conditions, and the lowest under methanogenic conditions. Rate constants for atrazine ranged from 1.04 × 10−5 to 3.32 × 10−5 L/mg VSS·h, although differences among the four electron acceptor conditions were not statistically significant.