Meeting report of the 4th European biotransformation workshop.

1. Challenges, strategies and new technologies in the field of biotransformation were presented and discussed at the fourth European Biotransformation Workshop which was held in collaboration with the joint ISSX/DMDG meeting on June 15, 2023 at the University of Hertfordshire in Hatfield, UK.2. In this meeting report we summarise the presentations and discussions from this workshop.3. The topics covered are listed below: Unusual biotransformation reactionsBiotransformation Workflows in Discovery utilising various softwares for structure elucidationBiotransformation software for the identification of peptide metabolitesAccelerator Mass Spectrometry (AMS) for endogenous and xenobiotic metabolite profilingMetabolite profiling using quantitative Nuclear magnetic resonance (NMR) and liquid chromatography coupled to inductively coupled plasma-mass spectrometry (LC-ICP-MS).

Meeting report of the 5th European Biotransformation Workshop.

Challenges, strategies and new technologies in the field of biotransformation were presented and discussed at the 5th European Biotransformation Workshop, which was held on March 14, 2024 on the Novartis Campus in Basel, Switzerland.In this meeting report we summarise the presentations and discussions from this workshop.The topics covered are listed below:Advances in understanding drug induced liver injury (DILI) risks of carboxylic acids and targeted covalent inhibitors.Biotransformation of oligonucleotide-based therapeutics including automated software tools for metabolite identification.Recent advances in metabolite synthesisQualification and validation of a new compact Low Energy Accelerator Mass Spectrometry (LEA) system for metabolite profiling.

Meeting report of the second European biotransformation workshop.

Challenges and opportunities in the field of biotransformation were presented and discussed at the 2nd European Biotransformation workshop which was conducted virtually in collaboration with the DMDG on November 24/25, 2021. Here we summarise the presentations and discussions from this workshop.The following topics were covered:Regulatory requirements and biotransformation studies for antibody drug conjugates (ADCs) and antisense oligonucleotides (ASOs).Solutions for mass spectral data processing of peptides and oligonucleotides.Future outsourcing needs in biotransformation for new modalities.Established quantitative and qualitative workflows for metabolite identification.New in vitro systems to study new chemical entities (NCEs) with low metabolic turnover.New strategies on the timing of the human ADME (absorption, distribution, metabolism, excretion) study and to investigate the impact of human microbiome on drug development.

Meeting report of the first European biotransformation workshop.

1. Challenges and opportunities in the field of biotransformation were presented and discussed at the 1st European Biotransformation workshop which was conducted virtually in collaboration with the DMDG 27 January 2021. Here we summarize the presentations and discussions from this workshop.The following topics were covered:2. Needs for radiolabel for IND filing versus quantitation without standards.3. Applications of cyclic ion mobility in the field of biotransformation.4. Computational predictions of xenobiotic metabolism.5. Future (outsourcing) needs in biotransformation.6. Genotoxicity risk assessment of metabolites and qualification of impurities using metabolite data.7. Regulatory aspects of MIST.

Crystal structures of ribonuclease F1 of Fusarium moniliforme in its free form and in complex with 2'GMP.

RNase F1, a guanine-specific ribonuclease from Fusarium moniliforme, was crystallized in two different forms, in the absence of an inhibitor and in the presence of 2'GMP. The crystal structure of the RNase F1 free form was solved by the molecular replacement method, using the co-ordinates of the RNase T1 complex with 2'GMP, and was refined to a final R-factor of 18.7%, using the data extended to 1.3 A resolution. For the crystal structure of the RNase F1 complex with 2'GMP, the solution of the molecular replacement method was obtained on the basis of the co-ordinates of the RNase F1 free form, and was refined to a final R-factor of 16.8%, using the data up to 2 A resolution. The two crystal structures of the RNase F1 free form and the complex with 2'GMP are very similar to each other as reflected by a small root-mean-square displacement (r.m.s.d.) value of 0.43 A for all C alpha atoms. The main differences between the two structures are associated with binding of 2'GMP in the substrate recognition site in the loop between Tyr42 and Glu46. A structural comparison between RNase F1 and RNase T1 shows a substantial similarity between all the C alpha atoms, as evidenced by a r.m.s.d. value of 1.4 A. The loop from residues 32 to 38 was strikingly different between these two enzymes, in both its conformation and its hydrogen bonding schemes. The side-chain of a catalytically active residue, His92, is shifted away from the catalytic site in RNase F1 by 1.3 A and 0.85 A with respect to the corresponding positions in the RNase T1 free form and in the RNase T1 complex with 2'GMP, respectively. In the RNase F1 complex, the guanine base of 2'GMP has a syn conformation about the glycosyl bond, and the furanose ring assumes a 3'-exo pucker, which is different from that found in the complex with RNase T1. In the catalytic site of the RNase F1 complex with 2'GMP, one water molecule was observed, which bridges the phosphate oxygen atoms of 2'GMP and the side-chains of the catalytically important residues, His92 and Arg77, through hydrogen bonds. A water molecule occupying the same position was found in the RNase F1 free form. The significance of this water molecule in the hydrolytic reaction is discussed.

X-ray studies on triclinic crystals of fatty acid binding protein. Examples of an extremely X-ray-resistant protein.

Fatty acid binding protein (pI 7.0) from bovine liver cytosol was crystallized using polyethylene glycol 4000 and 6000 as precipitating agents. The crystals are triclinic, space group P1. One molecule of 14 kDa occupies the unit cell with constants a = 33.5 A, b = 39.4 A, c = 30.6 A, alpha = 113.6 degrees, beta = 113.8 degrees, gamma = 88.8 degrees. Crystal diffraction extends to at least 2.25 A resolution and the crystals are stable in the X-ray beam for more than 450 h. One native data set to 2.5 A resolution has been collected.

Dose-dependent protein adduct formation in kidney, liver, and blood of rats and in human blood after perchloroethene inhalation.

Perchloroethene (PER) was a widely used solvent and is an environmental contaminant. In bioassays for carcinogenicity, PER was found to increase the incidence of liver tumors in mice and of renal tumors in male rats. Toxic effects of PER after repeated administration are likely caused by bioactivation. PER bioactivation occurs by two pathways. Oxidation by cytochrome P450 results in trichloroacetyl chloride, which binds to lipids and proteins. Glutathione S-conjugate formation from PER and further processing of the formed S-(trichlorovinyl)glutathione to S-(trichlorovinyl)-L-cysteine, followed by cysteine conjugate beta-lyase catalyzed cleavage, resulted in the reactive dichlorothioketene, which binds to proteins under formation of N epsilon-(dichloroacetyl)-L-lysine in proteins. The objective of this study was to comparatively quantify the dose-dependent formation of protein adducts from PER in rats and humans using antibodies with high specificity for either N epsilon-(trichloroacetyl)-L-lysine or N epsilon-(dichloroacetyl)-L-lysine in proteins. Male and female rats (n = 2, per concentration and time point) were exposed to 400, 40, and 10 ppm PER for 6 h and killed at various time points. Formation of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L-lysine in proteins was comparatively quantified in subcellular fractions from liver and kidney and in blood. In addition, three male and three female human volunteers were exposed to 10 and 40 ppm PER, and formation of protein adducts in blood was analyzed using the antibodies and GC/MS after immunoaffinity enrichment of modified proteins. In liver and kidney subcellular fractions and blood of PER-exposed rats, dose-dependent formation of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L-lysine in proteins was observed. Highest concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were formed in kidney mitochondria, followed by kidney cytosol. Only low concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were present in liver proteins; blood concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were 5 to 10 fold lower than in kidney mitochondria. Highest concentrations of N epsilon-(trichloroacetyl)-L-lysine were found in microsomal and cytosolic proteins from the liver of rats exposed to PER. A higher protein adduct formation was seen in PER-exposed-male than -female rats for N epsilon-(dichloroacetyl)-L-lysine in renal mitochondrial proteins, after exposure to 400 ppm PER. In human blood samples taken 0 and 24 h after the 6 h exposures to PER, N epsilon-(trichloroacetyl)-L-lysine-containing proteins were present in low concentrations. N epsilon-(Dichloroacetyl)-L-lysine-containing proteins were not detected either by Western blotting or GC/MS after immunoaffinity chromatography. The obtained results indicate a dose-dependent covalent binding of PER metabolites to proteins in rat liver, kidney, and blood and suggest that the concentration of covalent protein adducts is much lower in blood of humans as compared to the blood of rats exposed under identical conditions.

Ochratoxin A-induced tumor formation: is there a role of reactive ochratoxin A metabolites?

Ochratoxin A is a nephrotoxic and tumorigenic mycotoxin which contaminates a variety of food items, resulting in chronic human exposure. Biotransformation reactions have been implicated in the tumorigenicity of ochratoxin A. The biotransformation of ochratoxin A by cytochromes P450 and other mammalian enzymes was investigated to optimize conditions for bacterial mutagenicity testing. Metabolite formation was assessed by HPLC with UV and fluorescence detection and by LC/MS/MS. When ochratoxin A was incubated with liver microsomes from rats and mice, formation of 4R- and 4S-hydroxyochratoxin A was observed at very low rates. However, oxidation of ochratoxin A was not observed using kidney microsomes from rats and mice. Significantly higher rates of oxidation were seen in liver microsomes from rats pretreated with 3-methylcholanthrene and dexamethasone. Other reported or postulated that ochratoxin A-metabolites were not formed in detectable concentrations. Human cytochromes P450 (3A4, 1A2, and 2C9-1 Supersomes((R))) also showed very low activity with ochratoxin A (<60 fmole/min x pmol P450). Other enzyme systems used to study possible biotransformation of ochratoxin A were rat and human liver and kidney S-9 fortified with NADPH and glutathione, semipurified glutathione S-transferases, horseradish peroxidase, and soybean lipoxygenase; none of these resulted in detectable biotransformation of ochratoxin A. Using rat liver microsomes with high activity for ochratoxin A oxidation and the other enzyme systems to activate ochratoxin A for mutagenicity testing in the Ames test, mutagenicity was not observed in Salmonella typhimurium TA 100 and TA 2638. The obtained results suggest that oxidative biotransformation of ochratoxin A occurs at low rates, is catalyzed by cytochromes P450, and is unlikely to form reactive intermediates capable of binding to DNA.

Biotransformation and male rat-specific renal toxicity of diethyl ethyl- and dimethyl methylphosphonate.

Dimethyl methylphosphonate (DMMP) is a widely used chemical. Diethyl ethylphosphonate (DEEP) has been proposed as a replacement for DMMP in several applications. A long-term carcinogenesis study with DMMP in rats and mice showed a significant increase in the incidence of kidney tumors after 2 years of exposure in male but not in female rats and both sexes of mice. DMMP is not genotoxic. Due to these findings, a role of alpha(2u)-globulin accumulation in organ-specific tumorigenicity may be possible. alpha(2u)-Globulin is a low-molecular-weight protein synthesized in male rats under androgen control. Several male rat specific renal carcinogens have been shown to bind to alpha(2u)-globulin and to impair the renal degradation of this protein. This impairment results in alpha(2u)-globulin accumulation in the kidney, lysosomal overload, cell death, cell proliferation, and finally, renal tumor induction. To further characterize the toxicology of DMMP and DEEP, we investigated the biotransformation of these compounds and their ability to induce alpha(2u)-globulin accumulation in kidney. Biotransformation of both DMMP and DEEP were studied in male and female rats after single oral doses of 50 and 100 mg/kg. 31P-NMR and GC/MS showed that unchanged DMMP was excreted with urine; methyl phosphonate was identified as the only metabolite in urine. Unchanged DEEP was also excreted with urine; in addition, ethyl ethylphosphonate and ethylphosphonate were urinary metabolites. The majority of the applied dose of both compounds was recovered in urine within 24 h indicating rapid absorption and excretion. No sex-differences in rates of formation or excretion of metabolites were seen. To investigate alpha(2u)-globulin accumulation in the kidney after DMMP and DEEP, male and female Fischer-344 rats were administered DMMP or DEEP daily for five consecutive days by gavage. DMMP doses were 500- and 1,000-mg/kg body weight (bw); due to marked toxicity, daily DEEP dose of 50 and 100 mg/kg had to be used. Control rats received corn oil only and positive controls received five doses of 500-mg/kg bw trimethylpentane (TMP). Relative kidney weights were increased in male rats dosed with DMMP, DEEP, and TMP. alpha(2u)-Globulin in kidney cytosol was separated and quantified by capillary electrophoresis and by SDS-PAGE and Western blotting. In DMMP-, DEEP-, and TMP-treated rats, dose-dependent increases in the alpha(2u)-globulin content were observed by both methods in male, but not female rats. The increase of alpha(2u)-globulin accumulation was accompanied by the formation of protein droplets in the proximal tubules of male rats. These data demonstrate that the sex specific increase in kidney tumors by DMMP in male rats may be due to alpha(2u)-globulin accumulation and that similar toxic effects are to be expected from DEEP.

Quantitation of N epsilon-(dichloroacetyl)-L-lysine in proteins after perchloroethene exposure by gas chromatography-mass spectrometry using chemical ionization and negative ion detection followingimmunoaffinity chromatography.

An antibody specific to N epsilon-(dichloroacetyl)-L-lysine (DCA-Lys) was immobilized to immunoaffinity columns for the use in selective enrichment of dichloroacetylated proteins. These result from the reaction with dichlorothioketene the beta-lyase cleavage product of the perchloroethene metabolite S-(trichlorovinyl)-L-cysteine. Dichloroacetylated proteins from rat kidney mitochondria, rat plasma and human blood plasma were isolated after exposure to 40 ppm tetrachloroethene (PER) for 6 h. After acid hydrolysis of the protein fraction, DCA-Lys was derivatized with 1,3-dichloro-1,1,3,3-tetrafluoroacetone using N epsilon-(trifluoroacetyl)-L-lysine as internal standard. Recovery of dichloroacetylated reference proteins from immunoaffinity columns was about 73%. Samples were analyzed by GC-MS with chemical ionization and negative ion (NCI) detection showing DCA-Lys in proteins with 2.26 (+/- 0.02) pmol/mg protein in male rat kidney mitochondria and 1.92 (+/- 0.05) pmol/mg total mitochondrial protein in female rats. In rat plasma 0.47 (+/- 0.006) pmol DCA-Lys/mg protein in male and 0.34 (+/- 0.02) in female animals were found. DCA-Lys could not be detected in blood plasma of human volunteers exposed to PER with a detection limit of 20 fmol for the DCA-Lys derivative 2,2-bis(chlorodifluoromethyl)-4-(1-dichloroacetamido)-butyl- 1,3-oxazolidine-5-one. Immunoaffinity chromatography with specific antibodies provides a powerful tool for the enrichment of minor quantities of dichloroacetyled proteins in biological samples for GC-NCI-MS analysis of the modified amino acid lysine having broad utility in the biomonitoring of PER exposure.

Gas chromatography-negative ion chemical ionization mass spectrometry as a powerful tool for the detection of mercapturic acids and DNA and protein adducts as biomarkers of exposure to halogenated olefins.

The studies on metabolism of halogenated olefins presented here outline the advantages of modern mass spectrometry. The perchloroethene (PER) metabolite N-acetyl-S-(trichlorovinyl)-L-cysteine (N-ac-TCVC) is an important biomarker for the glutathione dependent biotransformation of PER. In urine of rats and humans exposed to PER, N-ac-TCVC was quantified as methyl ester after BF3-MeOH derivatization by gas chromatography with chemical ionization and negative ion detection mass spectrometry (GC-NCI-MS). The detection limit was 10 fmol/microliter injected solution using [2H3]N-ac-TCVC methyl ester as the stable isotope internal standard. Cleavage of S-(trichlorovinyl)-L-cysteine by beta-lyase enzymes results in an electrophilic and highly reactive thioketene which reacts with nucleophilic groups in DNA and proteins. Protein adduct formation was shown in kidney mitochondria by identification of dichloroacetylated lysine after derivatization with 1,1,3,3-tetrafluoro-1,3-dichloroacetone by GC-NCI-MS. In addition, chlorothioketene was generated in organic solvents and reacted with cytosine to give N4-chlorothioacetyl cytosine. After derivatization with pentafluorobenzyl bromide this compound exhibited good gas chromatographic properties and was detectable with a limit of detection of 50 fmol/injected volume. The detection of chemically induced protein modifications in the target organ of toxic metabolite formation and the study of DNA modifications with chemically generated metabolites provide important information on organ toxicity and possible tumorigenicity of halogenated olefins.

Neoantigen formation and clastogenic action of HCFC-123 and perchloroethylene in human MCL-5 cells.

In this study, the metabolic activation of 2,2-dichloro-1,1,1-trifluoroethane (hydrochlorofluorocarbons-123, HCFC-123), halothane or 1,1-dichloro-1-fluoroethane (HCFC-141b) was compared to that of perchloroethylene, using lymphoblastoma derived cell lines expressing human CYP1A1, CYP1A2, CYP2E1, CYP2A6 and CYP3A4 (MCL-5 cells). A dose dependent increase in micronucleus formation was detected over a nominal concentration range of 0.05-2 mM for HCFC-123 and halothane, but this was not seen with HCFC-141b. No dose response for HCFC-123 was seen in a control cHo1 cell line not expressing this cytochrome P450's. Cell lines expressing individual human cytochrome P-450 (CYP) forms were also used to define the enzymes responsible for the clastogenic events and to investigate the formation of immunoreactive protein by microsomal fractions. It was shown that CYP2E1 or CYP2B6 catalysed the clastogenic response, but CYP2D6, CYP3A4, CYP1A2 or CYP1A1 all appeared to be inactive. The formation of neoantigenic trifluoroacetylated protein adducts by microsomal mixtures incubated with HCFC-123 and NADPH was catalysed primarily by CYP2E1 and to a lesser extent by CYP2C19, whereas, only trace levels of immunoreactive protein were seen with microsomes expressing CYP2B6 or CYP2C8. With perchloroethylene as a substrate, the extent of activation was low in comparison with HCFC-123, as judged by the absence of micronuclei formation in the MCL-5 cell line and the weak immunoreactivity of proteins following Western blotting. CYP1A2, CYP2B6 and CYP2C8 appeared to be responsible for perchloroethylene immunoreactivity and in contrast to the findings with the HCFC's, no activation of perchloroethylene by CYP2E1 could be detected. These results show that even though both saturated and unsaturated halocarbons can result in neoantigen formation, there is a marked difference in the specificity of the CYP enzymes involved in their metabolic activation.

A probability representation for phase information from multiwavelength anomalous dispersion.

A probability distribution function, cast in the representation of Hendrickson & Lattman [Acta Cryst. (1970), B26, 136-143], has been derived for the phase information from measurements of multiwavelength anomalous diffraction (MAD). This probability function readily permits one to determine figure-of-merit weights similar to those used in isomorphous replacement, and the coefficients that characterize this distribution function facilitate the combining of MAD phasing with results from other sources of phase information. This probability representation was derived in the course of a structural analysis of selenobiotinyl streptavidin from MAD data and applications have also been made in the structure determinations of interleukin-1 alpha and a drug complex with brominated DNA.

Binding of hexachlorobutadiene to alpha 2u-globulin and its role in nephrotoxicity in rats.

Hexachlorobutadiene (HCBD) is nephrotoxic in rats causing damage to the proximal tubules. Renal toxicity is presumed to be due to bioactivation by glutathione S-conjugate formation and further processing by the enzymes of the mercapturic acid pathway to reactive intermediates. Recent studies revealed major sex-dependent differences in the pattern of urinary metabolites and gave evidence for the excretion of unmetabolized HCBD in the urine of male, but not female, rats. The objective of this study was to investigate the basis for the excretion of unchanged HCBD in the urine. We administered [14C]-HCBD (200 mg/kg bw, po) to male and female Sprague-Dawley (SD) and NCI Black-Reiter rats (NBR), an alpha 2u-globulin-deficient strain. No major differences in the disposition and in the rates of excretion of [14C]-derived radioactivity were observed between animals of both strains. Previously observed sex-specific differences in the formation of urinary metabolites in Wistar rats were now confirmed in SD rats and were also found in NBR rats. In contrast to male SD rats, however, NBR rats did not excrete unmetabolized HCBD with urine. [14C]-HCBD (10% of total urinary metabolites) was only present in the urine of male SD rats. Anion-exchange HPLC showed radioactivity associated with the alpha 2u-globulin fraction in urine and renal cytosol of male SD rats; the radioactive compound was identified as HCBD bound to the protein. The results indicate that the male-specific urinary excretion of HCBD is associated with its binding to alpha 2u-globulin. Light microscopic examination revealed the formation of hyaline droplets indicative of the accumulation of alpha 2u-globulin in the kidney of male SD rats after staining with Lee's methylene blue basic fuchsin. H&E staining additionally confirmed the finding of more pronounced necrotic changes in renal tubules of male SD rats than in females as previously described for Wistar rats. Binding of HCBD to alpha 2u-globulin may contribute to the pronounced nephrotoxicity in male rats.

Quantitation of alpha2u-globulin in rat kidney cytosol by capillary electrophoresis.

The renal accumulation of alpha2u-globulin has been implicated in the tumorigenicity of many nongenotoxic chemicals to the kidney of the male rat. Several chemicals inducing renal tumors in the male rat were shown to bind to alpha2u-globulin. This binding impairs the renal degradation of alpha2u-globulin, resulting in lysosomal overload, cell death, increased cell proliferation, and, presumably, renal tumor formation. To support the role of alpha2u-globulin accumulation in the renal toxicity of a chemical, a demonstration of the accumulation of this protein in the kidney of the male rat is one prerequisite. Monoclonal antibodies to alpha2u-globulin are available for quantifying alpha2u-globulin content; however, the procedure is time-consuming and complicated. We developed a method for the quantitation of alpha2u-globulin in renal cytosol using capillary electrophoresis. Renal cytosol fractions were analyzed by capillary electrophoresis as protein-SDS complexes. Using alpha2u-globulin purified from urine of male rats, the limit of detection was 10 microg/ml sample in routine analyses. Excellent run to run reproducibility in migration time (CV

Generation of antibodies to Di- and trichloroacetylated proteins and immunochemical detection of protein adducts in rats treated with perchloroethene.

Antibodies directed against chemical specific protein modifications are valuable tools to detect and comparatively quantify protein modifications. Both Nepsilon-(dichloroacetyl)-L-lysine and Nepsilon-(trichloroacety)l-L-lysine have been detected as modified amino acids in liver and kidneys of rats treated with perchloroethene (PER) after proteolysis. These protein modifications are formed by the interaction of reactive metabolites formed from PER with proteins. In this study we developed monospecific antibodies to dichloroacetylated and to trichloroacetylated amino acids to detect modified proteins in the target organs of PER toxicity. These antibodies were prepared by immunization of rabbits with modified keyhole limpet hemocyanin (KLH) coupled with either the dichloroacetyl or trichloroacetyl moiety. Enzyme-linked immunosorbent assays (ELISA) indicated that the polyclonal rabbit sera recognized dichloroacetylated or trichloroacetylated rabbit serum albumin (RSA), but not unmodified protein. Therefore, we further purified rabbit antisera on either Nepsilon-(dichloroacetyl)-L-lysine or Nepsilon-(trichloroacetyl)-L-lysine immobilized to immunoaffinity columns to obtain monospecific antibodies. The potential of these antibodies in the detection of di- and trichloroacetylated proteins and their selectivity for the desired dichloroacetyl or trichloroacetyl group was demonstrated in competitive enzme-linked immunosorbent assays with several structurally related compounds. Anti-dichloroacetyl (anti-DCA) antibody binding to dichloroacetylated RSA was inhibited by Nepsilon-(dichloroacetyl)-L-lysine with an IC50 value of 150 microM whereas inhibition by Nepsilon-(monochloroacetyl)-L-lysine and Nepsilon-(trichloroacetyl)-L-lysine showed an IC50 value of 100 mM. The binding of the anti-trichloroacetyl (anti-TCA) antibody to trichloroacetylated RSA was inhibited by Nepsilon-(dichloroacetyl)-L-lysine with an IC50 value of 80 mM. The inhibition by Nepsilon-(trichloroacetyl)-L-lysine was again 3 orders of magnitude stronger resulting in an IC50 value of 90 microM. Nepsilon-(acetyl)-L-lysine and unmodified RSA did not effect antibody binding to the chemically modified antigen. The antibodies were also successfully applied to detect modified proteins in subcellular fractions of liver and kidney from PER treated rats demonstrated in immunoblot. Protein adduct formation from different PER metabolism pathways was confirmed by the observation that the majority of dichloroacetylated proteins were located in kidney mitochondria and trichloroacetylated proteins were located in liver microsomes.

On the structure of crystalline ribulosebisphosphate carboxylase from Alcaligenes eutrophus.

Ribulosebisphosphate carboxylase from the hydrogen bacterium Alcaligenes eutrophus having a molecular weight of 534000 and consisting of eight large and eight small subunits has been crystallized by microdialysis using inorganic as well as organic precipitating agents. Crystals have tetragonal space group P42212, a = b = 11.27 nm, c = 20.14 nm, and contain one quarter molecule per asymmetric unit. X-rays are diffracted to 0.35-nm resolution on still photographs. Light optical diffractions of electron micrographs of thin sectioned crystals displayed patterns which could be interpreted on the basis of the unit cell determined by X-rays. Packing considerations are in accord with our earlier proposal regarding the subunit arrangement of this enzyme which differs from that reported for tobacco ribulosebisphosphate carboxylase.

Characterization and crystallization of core streptavidin.

We have characterized a streptavidin product that had been reduced to a minimal size that still retained full biotin-binding activity. This core streptavidin is proteolyzed at both ends at points that correspond closely with the termini of hen egg white avidin. Core streptavidin is more soluble than is the parent molecule. We have grown three different types of crystals of core streptavidin. The symmetry properties of these crystals prove that the molecule is a tetramer organized in tetrahedral (D2) point symmetry. The crystallographic response to the interaction of biotin with core streptavidin indicates that some conformational change accompanies ligand binding. We are attempting to determine the three-dimensional structure of streptavidin and its complex with selenobiotin from these crystals of core streptavidin.

Crystallization and preliminary X-ray investigation of avidin.

Hen egg-white avidin was crystallized at pH 6 from solutions containing 8-9% (w/v) poly(ethyleneglycol) 6000 as precipitant. X-ray investigation showed that the crystals are orthorhombic with cell constants a = 7.47 nm, b = 8.00 nm, c = 4.31 nm and belong to space group P21212. They contain half of a tetrameric molecule in the asymmetric unit.

Biotransformation of perchloroethene: dose-dependent excretion of trichloroacetic acid, dichloroacetic acid, and N-acetyl-S-(trichlorovinyl)-L-cysteine in rats and humans after inhalation.

Chronic exposure of rodents to perchloroethene (PER) increased the incidence of liver tumors in male mice and resulted in a small but significant increase in the incidence of renal tumors in male rats. The tumorigenicity of PER is mediated by metabolic activation reactions. PER is metabolized by cytochrome P450 and by conjugation with glutathione. Cytochrome P450 oxidation of PER results in trichloroacetyl chloride which reacts with water to trichloroacetic acid (TCA) which is excreted. The formation of S-(trichlorovinyl)glutathione (TCVG) from PER results in nephrotoxic metabolites. TCVG is cleaved to S-(trichlorovinyl)-L-cysteine (TCVC) and acetylated to N-acetyl-S-(trichlorovinyl)-L-cysteine (N-ac-TCVC), which is excreted with urine. TCVC is also cleaved in the kidney by cysteine conjugate beta-lyase to dichlorothioketene which may react with water to dichloroacetic acid (DCA) or with cellular macromolecules. The object of this study was to comparatively quantify the dose-dependent excretion of PER metabolites in urine of humans and rats after inhalation exposure. Three female and three male human volunteers and three female and three male rats were exposed to 10, 20, and 40 ppm PER for 6 h, and three female and three male rats to 400 ppm. A dose-dependent increase in the excretion of TCA and N-ac-TCVC after exposure to PER was found both in humans and in rats. A total of 20.4 +/- 7.77 mumol of TCA and 0.21 +/- 0.05 mumol of N-ac-TCVC were excreted in urine of human over 78 h after the start of exposure to 40 ppm PER; only traces of DCA were present. After identical exposure conditions, rats excreted 1.64 +/- 0.42 mumol of TCA, 0.006 +/- 0.002 mumol of N-ac-TCVC and 0.18 +/- 0.04 mumol of DCA. Excretion of N-ac-TCVC in male rats exposed to 400 ppm PER (103.7 nmol) was significantly higher, compared to female rats (31.5 nmol) exposed under identical conditions. N-ac-TCVC was rapidly eliminated with urine both in humans (t1/2 = 14.1 h) and in rats (t1/2 = 7.5 h). When comparing the urinary excretion of N-ac-TCVC, a potential marker for the formation of reactive intermediates in the kidney, humans received a significantly lower dose (3 nmol/kg at 40 ppm) compared to rats (23.0 nmol/kg) after identical exposure conditions. In addition, rats excreted large amounts of DCA which likely is a product of the beta-lyase-dependent metabolism of TCVC in the kidney. The obtained data suggest that glutathione conjugate formation and beta-lyase-dependent bioactivation of TCVC in PER metabolism is significantly higher in rats than in humans. Thus, using rat tumorigenicity data for human risk assessment of PER exposure may overestimate human tumor risks.