Doppler ultrasound for detection of renal transplant artery stenosis-threshold peak systolic velocity needs to be higher in a low-risk or surveillance population.

AIMS: To establish the ideal threshold arterial velocity for the diagnosis of renal transplant artery stenosis in a surveillance population with a low pre-test probability of stenosis. METHODS: Retrospective review of Doppler ultrasound, angiographic and clinical outcome data of patients transplanted over a 3-year period. Data used to calculate sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) for various threshold peak systolic velocity values. RESULTS: Of 144 patients transplanted, full data were available in 117 cases. Five cases had renal transplant artery stenosis-incidence 4.2% [stenosis identified at a mean of 6.5 months (range 2-10 months)]. All five cases had a significant arterial pressure gradient across the narrowing and underwent angioplasty. Threshold peak systolic velocity of > or =2.5 m/s is not ideal [specificity=79% (CI 65-82%), PPV=18% (CI 6-32%), NPV=100% (CI 94-100%)], subjecting many patients to unnecessary angiography-8/117 (6%) in our population. Comparable values if the threshold is set at > or =3.0 m/s are 93% (CI 77-96%), 33% (CI 7-44%) and 99% (CI 93-100%), respectively. The clinical outcome of all patients was satisfactory, with no unexplained graft failures or loss. CONCLUSIONS: In a surveillance population with a low pre-test probability of stenosis, absolute renal artery velocity > or =2.5 m/s is a limited surrogate marker for significant renal artery stenosis. The false-positive rate is high, and > or =3.0 m/s is a better choice which will halve the number of patients enduring unnecessary angiography. Close clinical follow-up of patients in the 2.5-3.0 m/s range, with repeat Doppler ultrasound if necessary, will identify the test false-negatives.

Variation in enzymes of arylamine procarcinogen biotransformation among bladder cancer patients and control subjects.

Arylamines such as 2-naphthylamine and 4-aminobiphenyl are suspected human bladder procarcinogens that require bioactivation to DNA-reactive species to exert their carcinogenic potential. The goals of the present study were (i) to assay for the presence of the arylamine acetyltransferases NAT1 and NAT2, and of the cytochrome P450 isoform CYP1A2, in human bladder epithelium; and (ii) to determine whether the activities of these arylamine biotransforming enzymes differ between bladder cancer patients and control subjects. We measured in-vitro enzyme activities in biopsies of normal, undiseased bladder epithelium obtained from 103 bladder cancer patients. NAT1 activity was detectable in all samples, with mean levels higher than those found in human liver. Kinetic evidence also suggested low levels of NAT2 expression in this tissue, but there was no detectable CYP1A2 by either enzymatic or immunochemical measurements. We also compared several probe drug indices of in-vivo NAT1, NAT2 and CYP1A2 activity between 53 bladder cancer patients and 96 cancer-free control subjects who were carefully matched for age, gender and smoking status. NAT1 and NAT2 genotypes were also determined. No significant differences were found between bladder cancer patients and control subjects for a number of individual phenotypic or genotypic predictors of enzyme function. Our results suggest that although expression of particular arylamine biotransforming enzymes within the bladder tissue could play a significant role in locally bioactivating arylamine procarcinogens in theory, interindividual variations in CYP1A2, NAT1 and NAT2 activities do not significantly differ between bladder cancer patients and control subjects when potential arylamine exposures are controlled for

The stability of thoracic segmentation in trilobites: a case study in developmental and ecological constraints.

The decline in origination rate of new metazoan body plans following the Cambrian radiation has been suggested to reflect developmental canalization in derived taxa, limiting their ability to evolve forms with radically different morphotypes. Segmentation is a fundamental aspect of arthropod body plan, and here we show that a derived trilobite that secondarily converged on a morphotype characteristic of basal members of the clade also reverted to a pattern of segmental variability common among basal trilobites. Hence a secular trend in loss of variability of the trilobite thorax was not due to the evolution of an inviolable developmental constraint. This result challenges the notion of developmental canalization in phylogenetically derived taxa. Rather, early variability in trilobites may be the result of ecological factors that promoted segment-rich thoracic morphotypes during Cambrian time.

Variants of N-acetyltransferase NAT1 and a case-control study of colorectal adenomas.

N-acetyltransferase NAT1, together with enzymes CYP1A2 and NAT2, helps convert heterocyclic amines to mutagens. Epidemiologic studies of the association of variants of these enzymes with colorectal cancer may provide indirect support for a heterocyclic amine mechanism. We used single strand conformation polymorphism and heteroduplex analysis to screen fro mutations in the NAT1 coding region in a case-control study (n = 932) of colorectal adenomas, which are precursors to cancer. Thirteen different single-base mutations were found: C97T, C190T, T402C, G445A-G459A-T640G ( a combination of three mutations), C559T, G560A, A613G, A752T, T777C, G781A, and A787G. Function of novel mutations was tested by bacterial production of enzymes and measurements of Km, Vmax, and stability. However, on 24-control individuals and 18 cases carried an inactivating NAT1 mutation. When combined with our data on the NAT2 acetylation polymorphism, we saw no evidence for an association between N-acetyltransferases and prevalence of adenomas. Larger sample sizes are required for further evaluation.

Structure, conformations, and repair of DNA adducts from dibenzo[a, l]pyrene: 32P-postlabeling and fluorescence studies.

The nature of stable DNA adducts derived from the very potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) in the presence of rat liver microsomes in vitro and in mouse skin in vivo has been studied using 32P-postlabeling and laser-based fluorescence techniques. Analysis of DB[a,l]P-DNA adducts via 32P-postlabeling has been obtained by comparison of the adduct patterns to those obtained from reactions of synthetic (+/-)-anti-, (+)-anti-, (-)-anti-, and (+/-)-syn-DB[a,l]P-11,12-diol 13,14-epoxide (DB[a,l]PDE) with single nucleotides and calf thymus DNA. anti-DB[a,l]PDE-dA adducts derived from the (-)-enantiomer are the major adducts formed in calf thymus DNA and in mouse skin DNA. The ratio of deoxyadenosine to deoxyguanosine modification is approximately 2:1 in mouse skin exposed to DB[a,l]P; activation by rat liver microsomes leads to a similar profile of adducts but with two additional spots. The conformations of DB[a,l]P adducts in native DNA, as well as the possibility of conformation-dependent repair, have been explored by low-temperature fluorescence spectroscopy. These studies have been performed using polynucleotides and calf thymus DNA reacted in vitro with DB[a,l]PDE and native DNA from mouse epidermis exposed to DB[a, l]P. The results show that adducts are heterogeneous, possess different structures, and adopt different conformations. External, external but base-stacked and intercalated adduct conformations are observed in calf thymus DNA and in mouse skin DNA samples. Differences in adduct repair rates are also revealed; namely, the analysis of mouse skin DNA samples obtained at 24 and 48 h after exposure to DB[a,l]P clearly shows that external adducts are repaired more efficiently than intercalated adducts. These results, taken together with those for B[a]P-DNA adducts [Suh et al. (1995) Carcinogenesis 16, 2561-2569], indicate that the repair of DNA damage resulting from PAH diol epoxides is conformation-dependent.

Identification and characterization of variant alleles of human acetyltransferase NAT1 with defective function using p-aminosalicylate as an in-vivo and in-vitro probe.

Although several variant alleles at the human NAT1 gene locus have been reported, their relationship to phenotypic variations in NAT1 function remains unclear. We have used in-vivo and invitro phenotyping tests, along with PCR-based cloning and heterologous expression, to investigate the extent of variation in NAT1 function and to characterize novel allelic variants at the NAT1 gene locus. The NAT1-selective substrate p-aminosalicylic acid (PAS) was used as a probe for NAT1 function. In-vivo PAS acetylation rates were estimated by determining the ratio of PAS to N-acetylated PAS (AcPAS) in urine and plasma following the oral ingestion of Nemasol Sodium. Excluding outliers, a 65-fold variation in the urinary AcPAS:PAS ratio was observed (n = 144), while a 5.6-fold variation in the plasma AcPAS:PAS ratio was seen in a subset (n = 19) of this sample. Urinary and plasma ratios correlated moderately (r = 0.74, p < 0.0005). One individual (case 244) had a marked impairment of PAS N-acetylation, with 10-fold lower urinary and plasma AcPAS:PAS ratios compared with other subjects. Biochemical investigations in whole blood lysates from case 244 suggested a NAT1 kinetic defect, with a 20-fold increased apparent K(m) for PAS and a 90-fold decreased Vmax for AcPAS formation. We subcloned, sequenced and expressed the protein-coding regions of the NAT1 alleles from case 244 and from seven other selected probands. Sequence analysis revealed the presence of two new variant alleles, designated as NAT1 x 14 and NAT1 x 15, in case 244, as well as one variant, NAT1 x 11, which has been observed in previous investigations. NAT1 x 14 contained a missense mutation (G560-->A) that is predicted to change a single amino acid (Arg187-->Gln), as well as two 3' non-coding region mutations (T1088-->A and C1095-->A) that have previously been observed in the NAT1 x 10 allelic variant. NAT1 x 15 had a single nonsense mutation (C559-->T; Arg187-->stop) and, thus, encodes a truncated protein. The activity of recombinant NAT1 14 mirrored the defective enzyme function in whole blood lysates from case 244, while NAT1 15 was completely inactive. Expressed NAT1 11, on the other hand, had identical activity to the wild type NAT1 4 allele, suggesting that the coding region mutations in this variant are functionally silent. The frequencies of NAT1 x 11, NAT1 x 14 and NAT1 x 15 were 0.021, 0.028 and 0.014 (n = 288 alleles), respectively, suggesting that they are relatively rare in our predominantly Caucasian sample.

Human acetyltransferase polymorphisms.

Conjugation of primary amino and hydroxylamino groups with acetate, catalyzed by acetyl CoA-dependent arylamine acetyltransferase (NAT) enzymes, may play an important role in the intricate series of metabolic pathways that produce or prevent toxicity following exposure to homo- and heterocyclic arylamine and hydrazine xenobiotics. Two independently regulated and kinetically distinct human acetyltransferases are now known to exist, namely NAT1 and NAT2. Interindividual variation in NAT2 function is associated with the classical isoniazid acetylation polymorphism which was discovered over forty years ago. At last count, fifteen variant alleles at the NAT2 gene locus have been linked to the isoniazid 'acetylator phenotype', and each of these can be identified in population studies using specific PCR-based genotyping tests. On the other hand, NAT1 shows kinetic selectivity for compounds whose disposition is unrelated to the classical isoniazid acetylation polymorphism. NAT1 expression is also phenotypically variable in human populations, at least in part due to allelic differences at the NAT1 gene locus. Nine NAT1 variant alleles have been described to date, of which NAT1* 14 and NAT1* 15 clearly produce defective NAT1 proteins and lead to functional impairment in the metabolism of NAT1-selective substrates both in vivo and in vitro. On the other hand, it has been reported that the NAT1* 10 variant associates with elevated NAT1 activity and increased risk for cancers of the bladder and colon. Because of the important toxicologic consequences of allelic variation in NAT1 and NAT2 function for the metabolic activation of arylamine and heterocyclic amine procarcinogens, further studies are needed to improve our understanding of the extent of NAT allelic variation, to determine the functional capacity of each variant gene product, and to develop accurate methods of detecting them in population and epidemiological studies.

Separation of 32P-labelled nucleoside 3',5'-bisphosphate adducts by HPLC.

Relatively few reported attempts have been made to substitute HPLC for the thin-layer ion-exchange chromatography (TLC) conventionally used in the 32P-postlabelling assay. Using a reverse-phase phenyl-modified silica gel column and a gradient of methanol in 0.5 M sodium phosphate buffer (pH 2.0), we were able to improve the resolution of very similar adducts. Combined with on-line detection of Cerenkov radiation, this method allows separation of sub-femtomole quantities of 32P-labelled nucleoside 3',5'-bisphosphates modified by bulky carcinogens. Using this method, we were able to separate nine of the ten major adducts formed by reaction of the diol-epoxides of ten polycyclic aromatic hydrocarbons with DNA, and resolve different adducts formed by a single carcinogen. The major adducts formed by benzo[b]fluoranthene (BbF) or dibenz[a,h]anthracene in mouse skin in vivo have been shown to be distinct from the adducts formed directly by the bay-region diol-epoxides. The heterocyclic amines IQ and MeIQ have each been shown to form one major DNA adduct in several in vitro and in vivo systems; using HPLC we were able to resolve the two adducts formed by these food mutagens. HPLC is especially useful for the identification of adducts by means of chromatographic comparisons and in the analysis of the multiple adducts formed by complex mixtures of environmental carcinogens. The major adducts formed by benzo[a]pyrene (BaP) and BbF in mouse skin in vivo that were not resolved on TLC were well separated by HPLC and thus a major DNA adduct formed in the skin of mice treated topically with coal tar was found to be derived from BaP rather than BbF.

32P-postlabelling analysis of the covalent binding of benzo[ghi]perylene to DNA in vivo and in vitro.

Benzo[ghi]perylene (B[ghi]P) is a polycyclic aromatic hydrocarbon (PAH), present in complex combustion products, and evidence for its carcinogenic activity in experimental animals is equivocal, and yet it has demonstrable mutagenic activity in vitro. In order to investigate the possible DNA binding properties of B[ghi]P, groups of male Parkes mice were treated topically with 1.0 mumol of B[ghi]P. Mice were killed up to 3 months after treatment, DNA was isolated from the treated areas of skin and analysed for adducts by 32P-postlabelling. Maximum levels of binding (0.57 fmol/microgram DNA) were detected 2 days after treatment and adducts were found to persist for at least 12 weeks after treatment, a property of many PAHs with known tumor-initiating activity. B[ghi]P also became bound to DNA in vitro in the presence of 3-methylcholanthrene-induced rat liver microsomal preparations. When chromatographed on PEI-cellulose, the major adducts formed by B[ghi]P in vivo and in vitro appeared to be identical. However, they were found to be different when compared by reversed-phase HPLC. These differences might explain, in part, the differences in the biological activity of B[ghi]P in vivo and in vitro. The behaviour of B[ghi]P when present in a mixture was also examined. B[ghi]P was applied topically to mouse skin with six other PAHs at a dose level of 0.25 mumol/PAH/mouse. DNA isolated 24 h after treatment was analysed for adducts by 32P-postlabelling. Whilst the total level of binding was 30% lower than expected from the sum of the binding levels that resulted when the hydrocarbons were applied singly, the formation of B[ghi]P-DNA adducts did not appear to be inhibited. The results have demonstrated that B[ghi]P has significant DNA binding ability in vivo and in vitro and on the basis of its DNA binding ability in mouse skin it would be predicted to be at least a weak tumour initiator. The formation of DNA adducts by B[ghi]P when present in an artificial mixture of PAHs suggest that B[ghi]P may contribute to the DNA binding activity of more complex carcinogenic mixtures.

Covalent binding of polycyclic aromatic hydrocarbon components of coal tar to DNA in mouse skin.

Treatment of mouse skin with coal tar is known to initiate tumour formation, with the carcinogenic activity associated mainly with polycyclic aromatic hydrocarbons (PAHs). A sample of pharmaceutical coal tar was analysed by gas chromatography and 19 major PAHs were identified. 32P-postlabelling analysis was used to characterize those PAHs that are responsible for the DNA binding of coal tar and, by inference, its biological activity. PAHs were grouped according to their reported carcinogenic activities and applied as mixtures to mouse skin. Group A contained all of the 19 PAHs, group B seven PAHs for which there is sufficient evidence for carcinogenicity and group C 12 PAHs with only limited or inadequate evidence of carcinogenicity in experimental animals. 32P-Labelled DNA adducts formed by coal tar were resolved on TLC into a pattern of three discrete spots (2, 4 and 6) and four areas of diffuse radioactivity (1, 3, 5 and 7). By comparison of the pattern of adducts formed by coal tar with those formed by the synthetic mixtures it appeared that PAHs in group B formed coal tar-DNA adduct spots 4 and 6, and that adduct spot 2 was formed by PAHs in group C. Attempts to identify those PAHs responsible for the formation of coal tar-DNA adducts 4 and 6 were made by comparing the chromatographic mobilities of 32P-labelled coal tar-derived DNA adducts formed in mouse skin, using TLC and HPLC, with those formed by PAHs in group B. As benzo[ghi]perylene (B[ghi]P), a component of group C, has been demonstrated to exhibit significant DNA binding ability previously, the chromatographic mobility of coal tar-DNA adduct spot 2 was compared to that of the major DNA adducts formed by B[ghi]P in vivo and in vitro. It appeared that coal tar adduct spot 2 was the major adduct formed by B[ghi]P in vitro and that benzo[a]pyrene, benzo[b]fluoranthene, benzo [j]fluoranthene and benzo[k]fluoranthene contributed to the formation of adduct spot 6. None of the PAHs examined appeared to be responsible for the formation of adduct spot 4.

Evidence of involvement of multiple sites of metabolism in the in vivo covalent binding of dibenzo[a,h]pyrene to DNA.

The in vivo formation of dibenzo[a,h]pyrene-DNA adducts in mouse skin was assessed by laser-excited fluorescence spectroscopy at 77 and 4.2 K. Two adducts were identified with fluorescence origin bands at approximately 383.5 and 407.2 nm, and these were shown to possess pyrene and benzo[a]pyrene (B[a]P) chromophores, respectively. Both DNA-bound chromophores displayed considerable electron-phonon coupling and likely assume a highly base-stacked or quasi-intercalated configuration within DNA duplexes. The presence of B[a]P and pyrene aromatic systems indicates that two-electron or monooxygenation metabolism occurred on either the a or h benzo moieties (which are equivalent) in the former case, and on both these rings in the latter case. The presence of two adduct species agrees with 32P-postlabeling analysis of the DNA, which showed the presence of two major adducts in both thin-layer and high-performance liquid chromatographic separations.

HPLC separation of 32P-postlabelled benzo[b]fluoranthene-DNA adducts.

Analysis using 32P-postlabelling and a recently developed HPLC method resolved the adduct formed by reaction of the benzo[b]fluoranthene (BbF) anti-bay-region diol-epoxide with DNA from the more polar major adduct produced by the hydrocarbon in three different biological systems. In each case, the adduct formed from the anti-bay-region diol-epoxide constituted only a minor proportion of the total DNA modification. Comparisons of the DNA adducts formed from the hydrocarbon with those formed in microsomal incubations from the putative metabolites BbF-9,10-diol, anti-BbF-9,10-diol-11,12-oxide and the 5,9,10- and 6,9,10-BbF-triols indicate that the predominant pathway for BbF activation in skin probably involves a bay-region triol-epoxide possessing a phenolic OH-group on the peninsula ring.

Covalent binding of dibenzpyrenes and benzo[a]pyrene to DNA: evidence for synergistic and inhibitory interactions when applied in combination to mouse skin.

Several well-documented examples of human exposure to carcinogens involve complex mixtures of polycyclic aromatic hydrocarbons (PAHs). Although the biological properties of many pure PAHs have been investigated, less is known about their effects when present as components of mixtures. As the ability to form DNA adducts in vivo is generally indicative of carcinogenic activity of PAHs, we have compared the DNA binding potencies of dibenzo[a,e]pyrene (DB[a,e]P), dibenzo[a,e]pyrene (DB[a,h]P), dibenzo[a,i]pyrene (DB[a,i]P), dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P), when applied topically, either singly or in combination, to the skin of male Parkes mice. DNA isolated from the skin and lungs was analysed by 32P-postlabelling. The adducts formed by each PAH exhibited markedly different chromatographic mobilities on polyethyleneimine-cellulose TLC plates. The relative binding potencies of the compounds in both skin and lungs were: DB[a,l]P much greater than B[a]P greater than DB[a,h]P greater than DB[a,i]P greater than DB[a,e]P, in good agreement with their reported carcinogenicities in mouse skin. The majority of adducts were removed from DNA within 21 days of treatment, but low levels of adducts were found to persist for at least 3 months in both tissues. When DB[a,l]P, DB[a,e]P and B[a]P were applied together to mouse skin, a total binding 31% lower than expected was detected, while with a mixture of DB[a,e]P and B[a]P the binding to DNA in skin was 65% higher than expected from the binding levels of the carcinogens when applied singly. Other binary combinations of these three PAHs gave adduct levels similar to the sum of the binding levels of the individual components when applied singly. The results demonstrate the usefulness of 32P-post-labelling for the assessment of the DNA binding potencies of PAHs in mouse tissues, and for the detection of interactions between components of mixtures of carcinogens.

Burns to infants using walking aids.

Thirty-one burns and scalds to infants using walking aids are recorded. The mechanism of injury and resulting morbidity are described. The need for more rigerous precautions is discussed.