Digitisation of the total burn surface area.

The assessment of surface area of the body affected by a burn (TBSA) has long been estimated with manual charts. Initial assessment of burned patients is made frequently by clinicians with limited experience producing significant errors. Paper copies of burn charts are unwieldy, subject to loss and tend towards overestimation. Thus, a simple method of calculation, recording and transmission via email or telemedicine may produce benefits in both initial treatment and data recording. Although computer-based systems have been reported previously none have entered routine clinical practice in the UK. We devised a PC-based program, "Burn Calculator", whereby digital transcription of the burn allows automatic area calculation allowing not only a rapid, accurate figure for determination of fluid resuscitation, but also the potential for rapid electronic transmission. It also calculates fluid requirements to minimise errors during resuscitation. This initial pilot study compared figures from 50 paper charts with those from Burn Calculator to determine its accuracy and reproducibility. Previously reported variations in TBSA estimation were confirmed, as was the tendency towards TBSA underestimation resulting from transcription of a three-dimensional clinical situation to a two-dimensional representation. Burn Calculator showed high correlation (r=0.9850; p<0.0001) and reproducibility (R=0.9957) that would simplify assessment and referral plus facilitate data collection, interpretation and research.

Genotoxicity and carcinogenicity studies of soy isoflavones.

The potential cancer preventive efficacy of soy isoflavones is being investigated in preclinical and phase 1 clinical studies sponsored by the U.S. National Cancer Institute. Although 90-day oral toxicity studies with PTI G-2535 (an investigational soy isoflavone drug product) in rats and dogs, as well as teratology studies, indicated no signs of toxicity, there remains a mechanistic concern associated with the ability of isoflavones (i.e., genistein) to inhibit topoisomerase, possibly leading to DNA strand breaks. The present report describes results from two in vitro genotoxicity studies, one in vivo genotoxicity study, and a single carcinogenicity study conducted in p53 knockout mice. Bacterial mutagenesis experiments using six tester strains without metabolic activation revealed no evidence that PTI G-2535 was mutagenic. In similar experiments with exogenous metabolic activation there were statistically significant increases in revertants, but less than twofold, in a single (Salmonella typhimurium TA100) tester strain. Mouse lymphoma cell mutagenesis experiments conducted with and without metabolic activation revealed statistically significant increases in mutation frequency at PTI G-2535 concentrations > or = 0.8 and 12 microg/ml, respectively; such increases were dose related and increases in the frequency of both small and large colonies were observed. A statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was also seen 24 hours after treatment in male, but not female, mice who received 500 and 1000 mg/kg body weight PTI G-2535; however,such increases were small, were not dose related, and were not observed 48 hours after treatment. In contrast, dietary genistein had no effect on survival, weight gain, or the incidence or types of tumors that developed in cancer-prone rodents lacking the p53 tumor suppressor gene, p53 knockout mice. The apparent risk/benefit of isoflavone ingestion may ultimately depend on the dose and developmental timing of exposure.

Variation in the promoter region of the myeloperoxidase gene is not directly related to lung cancer risk among male smokers in Finland.

In order to examine whether a polymorphism in the promoter region of the myeloperoxidase (MPO) gene is associated with lung cancer among male smokers, we conducted a case-control study nested within a Finnish clinical trial cohort. Although we found no evidence of an overall association between lung cancer risk and MPO genotype, the variant MPO genotype was associated with an increased risk of lung cancer among a subset of older men. These findings contrast with those from previous studies that report decreased lung cancer risk among MPO variant individuals.

Prognostic factors for hematologic cancers.

This article reviews the molecular biology of hematologic cancers and the current understanding of prognostic factors for these cancers. Specific molecular biomarkers that have potential as prognostic factors for various hematologic cancers are discussed. Quantitative and statistical methods of evaluating the usefulness of prognostic factors are presented.

Evaluation of the direct genotoxic potential of cadmium in four different rodent cell lines.

Cadmium is a toxic environmental contaminant that is carcinogenic in humans and laboratory animals. Although the mechanism underlying cadmium carcinogenesis has not yet been determined experimental evidence suggests that the stress-inducible, metal-binding proteins, metallothioneins, may mediate organ specificity. In the present study, four different rodent cell lines (Chinese hamster ovary cells, rat L6 myoblast cells, rat Clone 9 liver cells, and rat TRL 1215 liver cells) were exposed to 0, 1, 5, 10, 50, or 100 microM CdCl2 and monitored for evidence of direct DNA damage. A microfiltration assay was used to measure DNA strand breaks and a filter-binding assay was used to measure DNA-protein crosslinks, two lesions that have been associated with cadmium exposure and may mediate genotoxicity of the metal. Although variability in sensitivity to DNA damage was evident between the different cell lines, in all of the cell lines tested, increases in DNA damage were observed only at cadmium doses that completely arrested cell growth. In addition, in three of the four cell lines tested, induction of metallothionein had no substantial protective effect against cadmium-induced cytotoxicity or genotoxicty. While protection against cadmium-induced DNA strand breakage with metallothionein preinduction was observed in the TRL 1215 rat liver cells, metallothionein preinduction did not protect against cadmium-induced DNA-protein crosslinking in that cell line. Taken together, our results support the hypothesis that cadmium is not directly genotoxic.

Effect of cadmium exposure on background and anti-5 methylchrysene-1,2-dihydrodiol 3,4-epoxide-induced mutagenesis in the supF gene of pS189 in human Ad293 cells.

Cadmium is a toxic environmental contaminant that is carcinogenic in humans and rodents. Although cadmium has proven to be mutagenic in a variety of assay systems, exactly how cadmium achieves gentoxicity is poorly understood. To define the mechanism(s) underlying the mutagenicity and comutagenicity of cadmium, human Ad293 cells were exposed to subtoxic doses of the metal and transfected with untreated or anti-5-methylchrysene-3,4-dihydrodiol 1,2-epoxide (5-MCDE)-treated pS189 shuttle vector. Alterations in the frequency, types, and distribution of mutations were subsequently assessed in the supF gene of pS189 that was replicated in Ad293 cells and screened in indicator bacteria. Doses of 0.5 and 1 microM CdCl2 increased the mutation frequency of untreated pS189 by approximately 4- and 8-fold, respectively, with no apparent effect on the types of mutations generated. In contrast, host-cell exposure to cadmium had little or no effect on the frequency, types, or distribution of mutations generated with 5-MCDE-treated pS189. These results indicate that cadmium increases mutagenesis of untreated pS189 by affecting a process that is not involved in mutagenesis of the 5-MCDE-treated vector. Although it is not clear exactly how host-cell exposure to cadmium increases background mutagenesis, presumably, the mutagenic effect does not involve cadmium interaction with the cellular machinery used to replicate past bulky DNA lesions.

Lack of correlation between the inducibility of metallothionein mRNA and metallothionein protein in cadmium-exposed rodents.

Cadmium (Cd) is carcinogenic in humans and laboratory animals. Depending on the duration and route of exposure, Cd can also induce damage in the liver, kidneys and lungs. In certain tissues, metallothionein (MT) proteins are induced by Cd exposure and associated with native and acquired tolerance to the metal. Rats are generally more sensitive than mice to Cd carcinogenicity; however, sensitivity can vary markedly between different strains of the same rodent species. To further define the role of MT in Cd toxicity and carcinogenesis, adult male Wistar rats and adult male C57 and DBA mice were treated with CdCl2 and liver, kidney, and lung were analyzed for Cd, MT mRNA, and MT protein 24 h later. Dose-related increases in Cd were detected in the livers and kidneys of all animals tested; however, increases in pulmonary Cd were observed only in C57 mice, and only at the highest CdCl2 dose. While hepatic Cd concentrations were similar in the rats and mice, renal Cd concentrations were similar in the rats and DBA mice but were nearly 2-fold higher in C57 mice at the highest CdCl2 dose. Dose-related increases in MT mRNA occurred in the livers and lungs of all animals tested. Hepatic MT mRNA concentrations were highest in the rats, and C57 mice exhibited the greatest magnitude of hepatic MT mRNA induction. Dose-related increases in renal MT mRNA were also detected in both strains of mice, but between the two strains, C57 mice exhibited substantially higher levels of renal MT mRNA induction. Basal levels of renal MT mRNA were higher in the rats than in the mice, and transcription of the MT gene was not inducible in the rat kidney at any of the CdCl2 doses used. In comparison, basal levels of pulmonary MT mRNA were similar in the rats and DBA mice, were substantially lower in C57 mice, and increases in pulmonary MT mRNA were most pronounced in the rats. Analysis of MT protein revealed dose-related increases in the livers and kidneys of all animals tested. C57 mice had the lowest basal and induced levels of hepatic MT, and basal levels of renal MT were much higher in the rats than in mice of either strain. Although dose-related increases in pulmonary MT were similar in both strains of mice, pulmonary MT levels were much lower and not inducible in the rats. Overall our experiments revealed complex profiles of Cd distribution and MT expression that varied between tissues, species and strains, and often did not directly correlate with sensitivity to damage. The results suggest that Cd distribution, inducibility of the MT gene, and levels of MT protein, must all the considered when predicting susceptibility to Cd toxicity and carcinogenicity at particular target sites.

Evidence that nitric oxide enhances cadmium toxicity by displacing the metal from metallothionein.

Cadmium is carcinogenic in humans and rodents. Although extensive evidence indicates that the toxicity and genotoxicity of Cd is ameliorated by binding to cysteine clusters in metallothionein (MT), the factors governing Cd release at intracellular target sites remain unknown. Nitric oxide is a pollutant gas and an important intercellular messenger in the inflammatory immune response. When growing Chinese hamster ovary cells were treated for 24 h with 0.5, 0.75, or 1.0 mM CdCl2 followed by a 1-h exposure to 1.0, 1.5, or 2.0 mM 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), an NO-generating sodium salt, NO enhanced Cd-induced inhibition of colony forming ability without affecting Cd-induced cytolethality. In experiments designed to determine whether NO acts by displacing Cd from cellular MT, cells treated with 2.0 mM CdCl2 followed by 1.5 or 3.0 mM DEA/NO exhibited 29 and 38% reductions, respectively, in the amount of Cd bound to MT. When purified rat liver MT was used to further characterize NO-induced release of Cd from MT, dose-related increases in Cd displacement were observed at DEA/NO concentrations between 0.1 and 0.5 mM, and a plateau was reached at 3 mol of Cd displaced/mol of MT at higher DEA/NO concentrations. Compared to cells exposed to Cd or DEA/NO alone, cells treated with Cd followed by DEA/NO also exhibited a transient 2-3-fold decrease in c-myc proto-oncogene expression. Taken together, our results support the hypothesis that NO mediates Cd release from MT in vivo and suggest that intracellular generation of free Cd may induce DNA damage and force cells into a period of growth arrest. Such findings may have particular relevance with regard to the etiology of Cd-induced carcinogenesis in human populations.

A comparison of two ultrasensitive methods for measuring 1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-deoxycytidine in cellular DNA.

1,N6-Ethenodeoxyadenosine (edA) and 3,N4-ethenodeoxycytidine (edC) are two mutagenic adducts associated with exposure to ethyl carbamate (urethane) and vinyl chloride. We have recently developed two ultrasensitive methods for determining the molecular dose of these adducts in cellular DNA. In both methods, purified DNA was first enzymatically digested to 2c-deoxyribonucleotide 3c-monophosphates. Etheno-modified nucleotides were then separated from normal nucleotides in one of two ways: either by reverse phase, ion-pair HPLC coupled with 260 nm UV detection, or by immunoaffinity chromatography using reusable microcolumns containing specific monoclonal antibodies coupled to Protein A-Sepharose. Fractions enriched for the adducted nucleotides were labeled using T4 poly-nucleotide kinase and [32P]ATP, and individual nucleotides were subsequently resolved by two-dimensional TLC, visualized by autoradiography, and quantified by liquid scintillation counting. When used to analyze the same sample of etheno-modified calf thymus DNA, both assays produced similar results. However, when both methods were used to analyze rat liver DNA 'spiked' with known amounts of etheno nucleotide standards, the immuno-affinity/32P TLC procedure proved to be more sensitive and more reproducible than the HPLC/32P TLC method: while the detection limit of the immunoaffinity/32P TLC technique was < 4 etheno adducts/10(9) parent deoxynucleotides, the HPLC/32P TLC method often failed to detect adducts at concentrations < 2/10(8). In other experiments, the immunoaffinity/32P TLC method was used to demonstrate formation of edA and edC in cells treated with vinyl chloride monomer. Because of its exquisite sensitivity, the immunoaffinity/32P TLC method promises to be extremely useful for measuring both background and induced levels of etheno adducts, making it possible to examine the role of these adducts in inducing mutations and/or carcinogenesis.

Defective replication of psoralen adducts detected at the gene-specific level in xeroderma pigmentosum variant cells.

Replication of damaged DNA is suspected to play an important role in cell cycle, genetic stability, and survival pathways. Using psoralen photoaddition as prototype DNA damage and the renaturing agarose gel electrophoresis technique to measure DNA cross-linking in individual genes, Vos and Hanawalt previously observed efficient bypass replication of psoralen monoadducts in human genes (J.-M. H. Vos and P. C. Hanawalt, Cell 50:789-799, 1987). To understand the mechanism of bypass replication in human cells, mutants affected in such a process would be useful. We now report that cells from individuals suffering from the hereditary recessive syndrome xeroderma pigmentosum variant (XPV) are hypersensitive to killing induced by photoactivated psoralen. In addition, analysis of psoralen-mediated DNA cross-linking in the rRNA genes indicated that although repair of psoralen adducts was similar to that of normal individuals, XPV cells were markedly deficient in the ability to bypass psoralen adducts during replication; in comparison with normal cells, approximately half as many monoadducts were bypassed during replication in XPV cells. Furthermore, in contrast to normal cells, replication of interstrand cross-links was not detected in XPV. This is the first demonstration of a deficiency in bypass replication detected at the gene-specific level in vivo. A model involving a strand-specific defect in recombinational bypass in XPV is proposed.

Roles of dosage, pharmacokinetics, and cellular sensitivity to damage in the selective toxicity of cyclophosphamide towards B and T cells in development.

Cyclophosphamide (CP) is a known immunomodulating agent. When presented to either late stage chick embryos (e.g. 18 days of incubation (DI] or neonatal chicks. CP induces selective B cell damage resulting in humoral immunosuppression in chickens. The present study was undertaken in order to provide further insights into CP's selective immunotoxic effects. We investigated the influences of age, CP-dose, and drug distribution on CP-induced cytotoxicity in the B and T cell compartments of the developing chick. In this test system, differential immunotoxicity was strongly dosage-dependent; at all ages tested, B cell depletion predominated at low CP dosages (50 and 100 mg/kg) whereas equally extensive lymphocyte toxicity was observed in both thymus and bursa at 200 mg/kg drug levels. Furthermore, while immunotoxicity profiles were similar at the two later developmental timepoints (18 DI and 1 day post-hatch), significantly higher CP dosages were required to induce lymphoid damage at 12 DI. Pharmacokinetic studies with radiolabeled CP revealed that approximately two-fold higher levels of CP and its metabolites are taken up in bursal tissue as compared to the thymus. Experiments concerning the possible inherent differences in susceptibility to CP-induced mitotic inhibition and cell killing mediating selective toxicity towards B cells versus T cells showed that B cell mitosis was inhibited at CP dosages as low as 5 mg/kg. No such inhibition of T cell mitosis was observed at this same low dosage. However, mitosis was completely arrested in both B and T cells at 50 mg/kg CP. Observations of cellularity in immune organs shortly after CP exposure revealed that the bursa is at least ten times more sensitive than the thymus to CP-induced mitotic inhibition and killing of resident lymphocytes. These studies reveal that multiple factors are involved in modulating CP selective immunotoxicity in the developing embryo. These include the dosage level, preferential distribution of the drug to the bursa, and a much greater sensitivity of B cells to CP-mediated mitotic inhibition and cell killing.

Cyclophosphamide metabolism in the primary immune organs of the chick: assays of drug activation, P450 expression, and aldehyde dehydrogenase.

Several diagnostic catalytic assays were used to determine whether organ-specific metabolic activation or detoxification of cyclophosphamide (CP) contributes to the selective toxicity of CP directed towards differentiating B cells as compared to T cells in the developing chicken. An assay for the alkylation of 4-[p-nitrobenzyl] pyridine (NBP) was used to assess comparative levels of CP activation products generated from microsomal preparations from liver, bursa of Fabricius (B cells), and thymus (T cells) of day-old chicks. Three catalytic assays were used to characterize and compare cytochrome P450-associated enzyme activities in neonatal hepatic and lymphoid tissues. Aldrin epoxidase (AE) was used to detect phenobarbital (PB)-inducible P450 activity. Ethoxyresorufin-O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) were used for the evaluation of polycyclic aromatic hydrocarbon (PAH)-inducible P450 activities in control and PB- or 3,3',4,4'-tetrachlorobiphenyl (TCB)-induced animals. Using the NBP assay, basal and PB-induced CP activation were observed using chick liver microsomes. However, no evidence of CP activation from immune organ microsomes was observed in control, PB-, or TCB-induced chicks. Basal and PB-induced AE activities were observed in thymus, but not bursa, and represented less than 1% of basal liver activity. EROD activity was detected in TCB-induced samples from both thymus and bursa, the thymus having the greater activity. Activities of aldehyde dehydrogenase (ALDH), an enzyme involved in CP detoxification, were about equal in cytosolic fractions from the bursa and thymus. These studies suggest strongly that tissue-specific differences in metabolic capacities are not the major factors governing the selective toxicity of CP directed towards differentiating B lymphocytes in vivo.