Characterization of hamster recombinant monomorphic and polymorphic arylamine N-acetyltransferases: bioactivation and mechanism-based inactivation studies with N-hydroxy-2-acetylaminofluorene.

The purified hamster recombinant arylamine N-acetyltransferases (NATs), rNAT1-9 and rNAT2-70D, were characterized for their capabilities to bioactivate N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA binding reactants and for their relative susceptibilities to mechanism-based inactivation by N-OH-AAF. The rate of DNA adduct formation resulting from rNAT1-9 bioactivation of [14C]N-OH-AAF was more than 30 times greater than that of rNAT2-70D-catalyzed bioactivation of [14C]N-OH-AAF. This result is consistent with substrate specificity data indicating that N-OH-AAF is a much better acetyl donor for hamster NAT1 than NAT2. Previous studies indicated that N-OH-AAF is a mechanism-based inactivator of hamster and rat NAT1. In the presence of N-OH-AAF, both rNAT1-9 and rNAT2-70D underwent irreversible, time-dependent inactivation that exhibited pseudo first-order kinetics and was saturable at higher N-OH-AAF concentrations. The enzymes were partially protected from inactivation by the presence of cofactor and substrates. The limiting rate constants (ki) and dissociation constants (Ki) for inactivation by N-OH-AAF were determined. The second-order rate constants (ki/KI) were 22.1 min-1 mM-1 for rNAT1-9 and 1.0 min-l mM-1 for rNAT2-70D, indicating that rNAT1-9 is approximately 20 times more susceptible than rNAT2-70D to inactivation by N-OH-AAF. The kinetic parameters for rNAT1-9 were nearly identical to values previously reported for partially purified hamster NAT1. Partition ratios were 504 for inactivation of rNAT1-9 by N-OH-AAF and 137 for inactivation of rNAT2-70D. Thus, a turnover of almost 4 times as many N-OH-AAF molecules is required to inactivate each molecule of rNAT1-9 than is needed to inactivate rNAT2-70D. The partition ratio data are consistent with the finding that rNAT1-9 catalyzes a higher rate of DNA adduct formation by N-OH-AAF than rNAT2-70D. The combined results indicate that the recombinant enzymes are catalytically and functionally identical to hamster NATs and, therefore, will be a useful resource for studies requiring purified NATs.

Cyclic hydroxamic acid inhibitors of prostate cancer cell growth: selectivity and structure activity relationships.

BACKGROUND: Clinical symptoms of prostatitis, prostatodynia, and benign prostatic hyperplasia are relieved by the pollen extract cernilton, and the water-soluble fraction of this extract selectively inhibits growth of some prostate cancer cells. A cyclic hydroxamic acid, DIBOA, has been isolated from this extract and mimics its cell growth-inhibitory properties, but the specificity of DIBOA for inhibition of prostate cell growth has not been reported. METHODS: The in vitro growth inhibitory effects of DIBOA and nine structurally related compounds on DU-145 prostate cancer cells, MCF-7 breast cancer cells, and COS-7 monkey kidney cells were determined by treatment of the cells with various concentrations of the compounds for 2-6 days. RESULTS: The compounds exhibited a wide range of potencies, but none of them exhibited selective inhibition of DU-145 cell growth. MCF-7 cells were more sensitive to DIBOA than either DU-145 cells or COS-7 cells. 3,4-dihydroquinoline-2(1H)-one, compound (4), and 1-hydroxy-6-chloro-3,4-dihydroquinolin-2(1H)-one, compound (7), selectively inhibited MCF-7 cell growth at a concentration of 10 micrograms/ml. 1-hydroxy-3,4-dihydroquinolin-2(1H)-one, compound (3), and compound 7 were the most potent inhibitors of DU-145 cell growth. Treatment of DU-145 cells with 3 (100 micrograms/ml) substantially decreased the number of viable cells within 2 days, and no viable cells remained in the culture by day 4. CONCLUSIONS: It is unlikely that DIBOA, compound (1), is responsible for the selective growth inhibition of prostate cancer cells by the water-soluble fraction of the pollen extract cernilton. Cell morphology results indicate that the growth-inhibitory effects of DIBOA and structurally related agents on DU-145 cells are due to their ability to cause cell death.

Overexpression and large-scale purification of recombinant hamster polymorphic arylamine N-acetyltransferase as a dihydrofolate reductase fusion protein.

N-Acetyltransferases (NATs) are enzymes that catalyze the detoxification and/or bioactivation of a variety of xenobiotics. Rapid kinetic, biophysical, structural, and bioactivation studies on NATs require quantities of purified enzyme capable of being obtained only through recombinant DNA technology. This laboratory has previously developed a protein expression and purification system in which NATs are expressed as proteins fused to a FLAG octapeptide followed by a thrombin-cleavage site to allow liberation of the rNAT. Typically, however, only 0.5-1.5 mg of the recombinant NAT's could be readily purified in a single isolation sequence by immunoaffinity chromatography. Therefore, the expression system was modified by inserting the L54F dihydrofolate reductase (DHFR) mutant gene sequence between the FLAG octapeptide and the thrombin-cleavage site. Expression was carried out with TOPP3 Escherichia coli cells. The new purification methodology utilizes the unique pH dependence of binding to a methotrexate (MTX)-affinity column by the L54F DHFR mutant. Unfortunately, the affinity chromatography strategy did not work satisfactorily. Although the specific activity of the purified rNAT2 was comparable to that of NAT2 obtained from hamster tissue, only 3% of the activity was recovered. The apparent cause of the low recovery is the unanticipated irreversible binding of rNAT2 to MTX. Ion-exchange chromatography was investigated as an alternative purification method. An initial DEAE anion-exchange column resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin and reapplied to a DEAE anion-exchange column. The second DEAE column resulted in not only the separation of rNAT2-70D from FLAG-L54F DHFR, but also the purification of rNAT2-70D to near homogeneity. Application of the nearly homogeneous rNAT2-70D to a gel-filtration column resulted in recovery of homogeneous protein. The ion-exchange method of purifying rNAT2-70D is inexpensive and simple and yields more than 8 mg of pure enzyme from 1 liter of cell culture.

Heterocyclic N-acetoxyarylamines, models for the putative ultimate carcinogens of aromatic amines: 2-acetoxyamino-5-phenylpyridine and 2-acetoxyaminopyridine.

The structures of O-acetyl-N-(5-phenyl-2-pyridyl)-hydroxylamine, C13H12N2O2, (I), and O-acetyl-N-(2-pyridyl)hydroxylamine, C7H8N2O2. (II), have been determined in order to confirm earlier structure assignments based on spectroscopic information. Compound (I) is the probable mutagenic metabolite of the phenylalanine pyrolysis product 2-amino-5-phenyl-pyridine. The crystal structures of (I) and (II) are the first reported for heterocyclic N-acetoxyarylamines, the corresponding homocyclic arylamine derivatives being extremely unstable. In the solid state, both (I) and (II) exist as hydrogen-bonded dimers, with the arylamine N atom acting as donor and the pyridine N atom of a neighboring inversion-related molecule as acceptor; the distance between donor and acceptor N atoms is 3.007(2) in (I) and 2.956(2) A in (II). This orientation of the N-H bond results in the rotation of the acetoxy group out of the plane of the pyridine ring by 22.5(2) in (I) and 27.4(2) degrees in (II).

Arylamine N-acetyltransferases. Expression in Escherichia coli, purification, and substrate specificities of recombinant hamster monomorphic and polymorphic isozymes.

Two isozymes of arylamine N-acetyltransferases (NATs) catalyze the biotransformation of arylamines to arylamides, and the bioactivation of carcinogenic arylhydroxylamines and arylhydroxamic acids to reactive electrophiles capable of forming a variety of DNA and protein adducts. As part of a project directed toward delineation of the molecular factors responsible for the pronounced differences in the substrate specificity of the isozymes, we have recently reported the expression in Escherichia coli and purification of hamster NAT1 (NAT1 8) as a fusion protein to an antibody-reactive amino terminus FLAG peptide capable of being removed by digestion with enterokinase. Unfortunately, the conditions necessary for the removal of the peptide by enterokinase resulted in incomplete protease digestion and substantial loss of NAT1 activity. Consequently, we have constructed the plasmid pPH8 in which an 11 amino acid thrombin proteolysis site has been inserted between the FLAG peptide and the amino terminus of NAT1. In addition, a plasmid that expresses hamster NAT2 (NAT2 15) was constructed by exchanging the gene sequence for NAT1 with the cloned sequence for NAT2. Both NAT fusion proteins were expressed in JM105 cells. Analysis of catalytically active cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that approximately 30-fold more soluble NAT2 was expressed than NAT1 in the bacterial cytosol. The fusion proteins were purified to homogeneity by immunoaffinity chromatography, followed by gel filtration to remove high molecular weight contaminants. The FLAG peptide was subsequently removed by treatment with human thrombin, followed by an additional pass over the immunoaffinity column. Unlike the results obtained from proteolysis by enterokinase, the activities of the rNAT1 and rNAT2 were shown to be unaffected by treatment with thrombin. Moreover, the substrate specificities for the recombinant NATs closely matched those observed for NAT1 and NAT2 isolated from hamster liver.

Failure of oral taurine supplementation to influence skin-flap survival in rats.

Taurine was given orally to rats to determine its influence on survival of subdermal plexus skin flaps. Flaps were raised on the dorsum of 40 rats divided into groups given 0, 10, 30, or 100 mg/kg taurine daily starting 2 days before and continuing 14 days after surgery. No significant difference was found between groups for percentage of distal flap necrosis, although mean area of necrosis was less for the 30 mg/kg group. Oral taurine did not result in significant elevation of plasma taurine concentrations at day 16 of administration, although the 30 mg/kg group maintained a higher mean value. Analysis of skin taurine concentrations in flaps failed to detect significant differences between groups at each of three zones (proximal-normal, middle-transition, and distal-sloughed). All groups, except the 10 mg/kg group, had significantly lower taurine concentrations in the distal zone than in the normal and transition zones. In each group there was a trend toward lower taurine concentration from proximal to distal, suggesting that loss of tissue taurine may occur with tissue necrosis. Subjectively, no differences in skin histopathology were noted, but less severe skin lesions were more common in the 10 mg/kg group. Daily oral taurine supplementation rates of 10, 30, or 100 mg/kg appear not to significantly affect survival of subdermal plexus skin flaps in rats.

Hamster monomorphic arylamine N-acetyltransferase: expression in Escherichia coli and purification.

N-Acetylation is a major pathway in the metabolism of hydrazine and arylamine drugs, and has been associated with carcinogen bioactivation. Monomorphic hamster liver N-acetyltransferase (NAT1) cDNA was cloned from hamster liver cells by reverse transcriptase-coupled polymerase chain reaction. The determined nucleotide sequence was identical to that reported for NAT1. The NAT1 coding region was subcloned into the pG1 yeast expression vector, but cell extracts provided only transient acetyltransferase activity. In addition, cDNA was subcloned into the expression vectors pFLAG-ATS and pFLAG-MAC. The latter vectors encoded a tac promoter and appended a low-molecular weight (1 kDa) hydrophilic FLAG marker peptide to the amino terminus of NAT1. Unexpectedly, periplasmic export of FLAGATS-NAT1 by the ompA signal peptide of pFLAG-ATS proved to be detrimental to enzyme activity. High acetyltransferase activity, however, was obtained when the fusion protein was expressed in the cytosol. Enzyme purified to homogeneity by immunoaffinity chromatography exhibited substrate specificity comparable to that of the hamster-derived protein.

Inactivation of hamster monomorphic N-acetyltransferase by vinyl fluorenyl ketone.

Arylamine N-acetyltransferases (NATs) are cytosolic enzymes that play important roles in the detoxification and activation of xenobiotic arylamines and their metabolites. Vinyl fluorenyl ketone (VFK) is a selective and potent active site-directed irreversible inhibitor of rat liver monomorphic NAT. The present study demonstrated that VFK is an active site-directed affinity label for hamster liver monomorphic NAT, but is a much less effective inactivator of the polymorphic N-acetyltransferase isozyme. The potency, irreversibility and selectivity of VFK make it a potentially valuable tool for characterization of NATs that exhibit acetyl donor specificity similar to that of hamster monomorphic NAT.

Arylamine-nucleoside adduct formation: evidence for arylnitrene involvement in the reactions of an N-acetoxyarylamine.

N-Acetoxyarylamines are reactive metabolites that are produced from N-arylhydroxamic acids by N-arylhydroxamic acid N,O-acyltransferase and by the acetyl coenzyme A-dependent O-acetylation of N-arylhydroxylamines. Solvolytic decomposition of 7-acetyl-2-(N-acetoxyamino)fluorene (3) afforded 7-acetyl-2-aminofluorene (4) and 7,7'-diacetyl-2,2'-azofluorene (5). Solvolysis of 3 in the presence of 2'-deoxyguanosine resulted in formation of N-(deoxyguanosin-8-yl)-7-acetyl-2-aminofluorene (2), along with smaller yields of 4 and 5 than were obtained in the absence of 2'-deoxyguanosine. The inclusion of nitrene trapping agents, such as piperylene, triethyl phosphite, 1-chloro-4-nitrosobenzene, and oxygen, in the reaction medium with 3 and 2'-deoxyguanosine caused a reduction in the yields of 2, 4, and 5. Additionally, products that would be expected to be formed through reaction with an arylnitrene intermediate were obtained when triethyl phosphite, 1-chloro-4-nitrosobenzene, and oxygen were included in the incubations with 3 and 2'-deoxyguanosine. The results support the proposal that a nitrene intermediate is involved in the formation of 2, 4, and 5 from 3.

Bioactivation of N-hydroxy-2-acetylaminofluorenes by N,O-acyltransferase: substituent effects on covalent binding to DNA.

N-Acetoxyarylamines are reactive metabolites that lead to arylamine adduct formation with biological macromolecules. A series of 7-substituted-N-hydroxy-2-acetylaminofluorenes were converted to reactive N-acetoxyarylamines by enzymatic N,O-acyltransfer in the presence of DNA. The N-arylhydroxamic acid substrates that contained electronegative 7-substituents formed greater amounts of DNA adducts than either the unsubstituted compound (N-OH-AAF) or those analogs that contained electron-donating groups in the 7-position. Glutathione did not decrease the rates of DNA adduct formation, but other nucleophiles, such as potassium O-ethylxanthate, thiourea and N-acetylmethionine, inhibited adduct formation by the 7-Br-substituted compound (7-Br-N-OH-AAF) and the unsubstituted parent compound (N-OH-AAF). Nucleophiles, reducing agents (e.g. ascorbic acid) and spin-trapping agents had minimal effect on DNA adduct formation by the bioactivated form of 7-acetyl-2-(N-hydroxy-acetylamino)fluorene (7-Ac-N-OH-AAF). Triethylphosphite, an agent that reacts with aryl nitrenes, caused a concentration-dependent reduction in the amount of DNA adduct formed subsequent to bioactivation of 7-Ac-N-OH-AAF, but did not influence adduct formation when N-OH-AAF and 7-Br-N-OH-AAF were the substrates. The results indicate that a change in the reaction mechanism(s) responsible for DNA adduct formation occurred when the strongly electronegative acetyl group was incorporated into the 7-position of N-OH-AAF. It is proposed that a nitrene intermediate is involved in the formation of covalent adducts with DNA when 7-Ac-N-OH-AAF is activated by N,O-acyltransfer.

Evaluation of rapid gross visual appraisal of swine lungs at slaughter as a diagnostic screen for enzootic pneumonia.

A rapid gross visual appraisal of enzootic pneumonia lesions was made of 87 lungs at a local abattoir and the lungs were then set aside and examined in more detail. A sample of pulmonary tissue was taken from each lung and submitted for bacterial and histological examination. The principal investigator who had performed the gross and detailed lung scores was then used to assess the agreement of two inspectors who were scoring lung lesions in the abattoir. The Pearson's correlation coefficient between grossly scored lungs and scores derived from a detailed examination was 0.94. Using histological examination as the gold standard, rapid gross examination had a sensitivity of 76% and a specificity of 71%. Using bacterial recovery as a gold standard yielded a sensitivity of 77% and a specificity of 51%. The sensitivity and specificity of inspectors 1 and 2 compared to the principal investigator were: sensitivity = 97.5% and specificity = 97.4% for inspector 1, and 97% and 98% for inspector 2. The kappa values for both of the inspectors compared to the principal investigator were 0.95 suggesting that designated lay inspectors consistently agreed with the principal investigator.

Comparative toxicity and mutagenicity of N-hydroxy-2-acetylaminofluorene and 7-acetyl-N-hydroxy-2-acetylaminofluorene in human lymphoblasts.

Exponentially growing TK6 human lymphoblasts were exposed to either 0-50 microM N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or 0-10 microM 7-acetyl-N-hydroxy-2-acetylaminofluorene (7-acetyl-N-OH-AAF) in both the absence and presence of a partially purified preparation of hamster-liver N-arylhydroxamic acid N,O-acyltransferase (AHAT). Neither N-arylhydroxamic acid was toxic to the lymphoblasts, nor mutagenic at the thymidine kinase (tk) locus, in the absence of AHAT over the concentration range examined. In the presence of AHAT, an enzyme that activates N-arylhydroxamic acids to electrophilic N-acetoxyarylamine intermediates, both compounds caused toxicity and mutagenicity in TK6 cells. The 7-acetyl-N-OH-AAF was approximately 10-fold more toxic and mutagenic than the unsubstituted N-OH-AAF. These data demonstrate that metabolism of these N-arylhydroxamic acids, presumably to N-acetoxyarylamine intermediates by AHAT, is a key event in the biological activity of these agents. In addition, the presence of electron-withdrawing 7-acetyl substituent that is thought to stabilize N-acetoxy intermediates, appears to enhance the biological activity of the unsubstituted N-OH-AAF.

Affinity alkylation of hamster hepatic arylamine N-acetyltransferases: isolation of a modified cysteine residue.

N-Acetyltransferases (NATs) play key roles in the detoxification and/or bioactivation of arylamines, arylhydroxylamines, arylhydroxamic acids, and hydrazines in mammalian tissues. In the present study, two hamster hepatic NATs (NAT I and NAT II) were separated, and each was purified greater than 2000-fold by sequential ammonium sulfate fractionation, DEAE anion exchange chromatography, Sephadex G-75 gel filtration chromatography, aminoazobenzene-coupled affinity chromatography, and DEAE anion exchange high performance liquid chromatography. Both NAT I and NAT II were purified to near-homogeneity. The molecular masses of NAT I and NAT II were estimated to be 30.5 kDa and 32.6 kDa, respectively. 2-(Bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide were synthesized and evaluated as affinity labels for NAT I and NAT II. Whereas Br-AAF was a highly selective inactivator of NAT II, bromoacetanilide inactivated both NAT I and NAT II in a similar fashion. Inactivation of NAT II by both Br-AAF and bromoacetanilide, and inactivation of NAT I by bromoacetanilide, followed pseudo-first-order kinetics. Relative rate constants (k(obs)/[I]) for the two compounds indicate that Br-AAF is approximately 25 times more potent than bromoacetanilide as an inactivator of NAT II. Both acetylcoenzyme A (CoASAc) and 2-acetylaminofluorene protected NAT II from inactivation by Br-AAF, and CoASAc provided protection of both NAT I and NAT II activities from inactivation by bromoacetanilide, indicating that the inactivation by both bromoacetanilide and Br-AAF is active site directed. The irreversibility of the inactivation of NATs by Br-AAF and bromoacetanilide was demonstrated by the failure to recover transacetylase activities after gel filtration of enzyme preparations that had been preincubated with Br-AAF or bromoacetanilide. Preincubation of NAT II with CoASAc significantly reduced the incorporation of [14C]Br-AAF into the enzyme, providing further evidence that the labeling is active site directed. In addition, pretreatment of NAT II with N-ethylmaleimide completely prevented the labeling of NAT II with [14C]Br-AAF, which suggests that a cysteine thiol is the target nucleophile of Br-AAF. High performance liquid chromatography analysis of the hydrochloric acid hydrolysate of [14C]Br-AAF-labeled NAT II revealed that 70% of total radioactivity is associated with S-carboxymethyl-L-cysteine, indicating that Br-AAF reacts primarily with a cysteine residue at the active site. These studies provide direct evidence that hamster hepatic NAT II contains an essential cysteine residue at the active site, and they establish the potential utility of Br-AAF for determining amino acid sequences in the active site of hamster hepatic NAT II.

Effect of group-selective modification reagents on arylamine N-acetyltransferase activities.

Two forms of hamster hepatic arylamine N-acetyltransferase (NAT; EC 2.3.1.5), designated NAT I and NAT II, were purified 200- to 300-fold by sequential 35-50% ammonium sulfate fractionation, Sephadex G-100 gel filtration chromatography, AAB affinity chromatography, DEAE ion exchange chromatography, and P-200 gel filtration chromatography. Treatment of either NAT I or NAT II with N-ethylmaleimide (NEM), a cysteine selective reagent, caused a concentration-dependent loss of enzymatic activities. Acetyl coenzyme A (AcCoA) protected NAT I against inactivation by NEM, whereas both 2-acetylaminofluorene (2-AAF) and AcCoA protected NAT II against inactivation. Incubation of either NAT I or NAT II with phenylglyoxal (PG), an arginine selective reagent, caused a time-dependent and a concentration-dependent loss of both NAT I and NAT II activities; the inactivations followed pseudo first-order kinetics. The reaction order with respect to PG was approximately two for each enzyme, consistent with the expected stoichiometry for the reaction of PG with arginine. The presence of AcCoA provided full protection of NAT I against inactivation by PG. However, neither AcCoA nor 2-AAF provided protection of NAT II against inactivation by PG. Diethylpyrocarbonate (DEPC), a histidine selective reagent, caused time-dependent and concentration-dependent pseudo first-order inactivation of both NAT I and NAT II. Neither AcCoA nor products of NAT-catalyzed reactions protected NAT I and NAT II against inactivation by DEPC. These results suggest that cysteine, arginine and histidine residues are essential to the catalytic activity of both NAT I and NAT II; the cysteine(s) is located at or near the binding site of NAT I and NAT II, and the arginine residue appears to be located in the AcCoA binding site of NAT I. In contrast, the essential arginine residue(s) of NAT II and the essential histidine residue(s) of both NAT I and NAT II are not likely to reside in the binding site of the enzymes.

Surgical treatment of a congenital bronchoesophageal fistula in a dog.

A 1-year-old male Cairn Terrier was evaluated for chronic coughing that was aggravated by eating or drinking. Radiography revealed an esophageal diverticulum, regional megaesophagus, and focal interstitial densities in the right caudal and middle lung lobes. Using fluoroscopy and contrast radiography, contrast material was seen to accumulate in the diverticulum and to reflux into the right middle, caudal, and accessory bronchi. Radiographic diagnosis was bronchoesophageal fistula. Via right eighth intercostal space thoracotomy, the abnormal connection between esophagus and caudal lobe of the right lung was identified, the lobe was resected, and the esophagus was closed. Histologic examination of the connecting tissue revealed a lining of stratified epithelium, with the superficial layer being predominantly ciliated columnar epithelium. Several findings led to the conclusion that the fistula was a congenital lesion, arising from aberrant formation of the respiratory tract from the embryologic digestive tract. Histologic examination revealed smooth muscle and lack of inflammation in tissue surrounding the fistula, which are criteria for identifying congenital bronchoesophageal fistula in human patients. The dog was young and did not have a history of esophageal foreign bodies. Postoperative complications were not encountered, and 9 months later, the dog was reported to be eating dry dog food without coughing. Congenital and acquired bronchoesophageal fistulas in dogs are reported infrequently. Furthermore, 2 of 12 previously reported bronchoesophageal fistulas in dogs, one of which was considered congenital, developed in Cairn Terriers.

Postmortem eyefluid analysis in dogs, cats and cattle as an estimate of antemortem serum chemistry profiles.

This study was carried out to determine the diagnostic usefulness of postmortem eyefluid analysis in estimating antemortem concentrations of serochemical constituents. A total of 31 cattle, 18 dogs and 22 cats were selected from routine elective euthanasia submissions to a diagnostic laboratory. For all cases, a biochemical profile, including determinations for electrolytes, glucose, urea, creatinine, enzymes, cholesterol, bilirubin, protein and osmolality was performed on antemortem serum, and postmortem aqueous and vitreous humors at 0 and 24 h incubation periods. The association between serum and postmortem eyefluid chemistry values was examined using simple linear regression. A strong correlation between serum and postmortem eyefluid urea and creatinine concentrations was demonstrated in the three species examined over a 24 h postmortem interval. We concluded that an accurate estimate of antemortem serum urea or creatinine can be made from the analysis of aqueous or vitreous fluid at necropsy. An estimation of antemortem serum electrolytes (including calcium in cattle) cannot be made with a high degree of accuracy due to the amount of variability in the relationship between serum and eyefluid electrolyte values. For large molecules such as proteins, enzymes, cholesterol and bilirubin there was very poor correlation between serum and eyefluid values.

An isozyme-selective affinity label for rat hepatic acetyltransferases.

Affinity chromatography of an ammonium sulfate precipitate obtained from rat hepatic cytosol resulted in the separation of two fractions of N-acetyltransferase (NAT) activity. NATI catalyzed the S-acetylcoenzyme A (AcCoA)-dependent acetylation of p-aminobenzoic acid (PABA); NAT II catalyzed the N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-dependent acetylation of 4-amino-azobenzene (AAB) (N,N-acetyltransferase), the AcCoA-dependent acetylation of procainamide (PA), and the N-arylhydroxamic acid N,O-acyltransferase (AHAT) activity that results in the conversion of N-OH-AAF and related hydroxamic acids to electrophilic reactants. 1-(Fluoren-2-yl)-2-propen-1-one (vinyl fluorenyl ketone, VFK) was shown to be a potent and irreversible inactivator of NAT II activities. A 200-fold higher concentration of VFK was required to inactivate NAT I activity than was required for inactivation of NAT II activities. Similar selectivity in the inactivation of the isozymes was observed when experiments were conducted with enzyme preparations that contained both NAT I and NAT II activities. The presence of substrates and products of the NAT II-catalyzed reactions such as AcCoA, 2-acetylaminofluorene (2-AAF), and N-acetyl-4-aminoazobenzene (N-Ac-AAB) protected NAT II from the inactivating effects of VFK, providing evidence that VFK is an active site directed inhibitor (affinity label) of NAT II. Studies with 1-(fluoren-2-yl)-2-propan-1-one (EFK), an analogue of VFK in which the alpha, beta-unsaturated vinyl ketone group of VFK has been replaced with an ethyl ketone group, demonstrated that the conjugated ketone of VFK is required for inactivation of enzyme activity. The results of these studies suggest that agents such as VFK should have utility as probes of acetyltransferase multiplicity and in the investigation of the active site topography of the enzymes.

Analogues of N-hydroxy-4-acetylaminobiphenyl as substrates and inactivators of hamster hepatic acetyltransferases.

1. A series of analogues of N-hydroxy-4-acetylaminobiphenyl (1, N-OH-AAB) has been synthesized and evaluated in vitro as substrates and inactivators of hamster hepatic N,N-acetyltransferase (N,N-AT) activity. The analogues of 1 are N-arylhydroxamic acids in which an atom or small functional group has been incorporated between the phenyl rings of 1. 2. The structural and molecular properties of the atoms between the two phenyl rings had little influence on the ability of the compounds to serve as acetyl donors in the N-arylhydroxamic acid-dependent transacetylation of 4-aminoazobenzene (AAB) catalysed by N,N-AT. An exception was the SO2 analogue (6) which was inactive. 3. All of the compounds except 6 were mechanism-based inactivators (suicide inhibitors) of hamster hepatic N,N-AT. The inhibition of N,N-AT by the hydroxamic acids was irreversible. The properties of the atom or functional group between the phenyl rings had a substantial influence on the relative effectiveness of the compounds as inactivators of N,N-AT. trans-N-Hydroxy-4-acetylaminostilbene (N-OH-AAS, 7) was the most potent and effective mechanism-based inactivator among the compounds studied. The ketone analogue (2) was the least effective among the compounds that exhibited inactivating activity. 4. The presence of the nucleophile cysteine in the incubation mixtures reduced the extent of inactivation of N,N-AT by 1 and by the ether (4) analogue but had little effect on the inactivation caused by 7. The inactivation of N,N-AT by N-OH-AAS (7) does not appear to involve electrophiles that are released from the active site and subsequently become covalently bound to the enzyme.