Barbara C. Levin

Forensic applications of mitochondrial DNA

Human mitochondrial DNA has become a useful tool in forensic investigations. Its polymorphic nature and maternal inheritance are characteristics that have, combined with its sequence information, enabled investigators to identify missing persons, war casualties and individuals involved in mass disasters and criminal cases. Various screening procedures have been developed to reduce the need to sequence samples that do not match, but DNA-sequence information is still necessary to verify a match. Even though several challenges remain before mitochondrial-DNA-sequence information can be used unambiguously, comparative mitochondrial-DNA-sequence analysis appears to be a reliable and powerful means for human identification.

Effects of exposure to single or multiple combinations of the predominant toxic gases and low oxygen atmospheres produced in fires

The toxicity of single and multiple fire gases is studied to determine whether the toxic effects of the combustion products from materials can be explained by the toxicological interactions (as indicated by lethality) of the primary fire gases or if minor, more obscure gases need to be considered. LC50 values for Fischer-344 rats have been calculated for the individual gases, carbon monoxide (CO), hydrogen cyanide (HCN), or decreased oxygen (O2), for 30-min exposures plus relevant postexposure periods using the NBS Toxicity Test Method. Combination experiments with CO and HCN indicate that they act in an additive manner. Synergistic effects have been found when the animals are exposed to certain combinations of CO and carbon dioxide (CO2). Five percent CO2 raised the threshold for deaths due to hypoxia and decreased the LC50 of HCN. Decreasing the O2 concentration in the presence of various mixtures of the other major fire gases increased the toxicity even further. A comparison of the concentrations of the major combustion products generated from a number of polymeric materials at their LC50 (30-min exposure plus 14-day postexposure) values with the combined pure gas results indicates that, in most cases, the observed toxicity may be explained by the toxicological interactions of the examined primary toxic fire gases. These results provide necessary information for the computer model currently being developed at the Center for Fire Research to predict the toxic hazard that people will experience under various fire scenarios.

A methodology for obtaining and using toxic potency data for fire hazard analysis

A comprehensive methodology has been developed for obtaining and using smoke toxicity data for fire hazard analysis. This description of the methodology comprises (1) determination that the post-flashover fire is the proper focus of smoke inhalation deaths; criteria for a useful bench-scale toxic potency (LC50) measurement method; (2) a method which meets these criteria, especially validation against real-scale fires; (3) a computational procedure for correcting the results from the bench-scale test for the CO levels observed in real-scale post-flashover fires; (4) procedures for reducing the usage of animals and broadening the applicability of data by interpreting gas measurement data using the N-Gas Model; and (5) a procedure for identifying whether a product produces smoke within the ordinary range of toxic potency for postflashover fires.

Toxic potency measurement for fire hazard analysis

SummaryThis report is the principal product of a long-term research program to provide a technically sound methodology for obtaining and using smoke toxicity data for hazard analysis. It establishes:(a)an improved bench-scale toxic potency1 measurement, one which represents the important combustion conditions of real fires; and(b)a design and analysis framework which will allow the toxic potency data to be used in a rational, consistent, appropriate, and adequate way. This establishment of proper bench-scale test conditions, validation of the output against real-scale fire measurements, and development of a consistent framework for the inclusion of toxic potency in fire hazard2 analysis is unique and represents a successful, usable implementation of the state of the art.This method focuses on post-flashover fires. The U.S. fire statistics show that 69% of all fire deaths are associated with post-flashover fires, with the preponderance of deaths due to smoke inhalation and occurring outside the room of fire origin. These fires are characterized by:• primarily radiant heating, with heat fluxes from about 20 to 150 kW/m2 throughout the room;• many items simultaneously on fire; and• vitiated combustion air for some, but not all, burning items.

Analysis of carboxyhemoglobin and cyanide in blood from victims of the Dupont Plaza Hotel fire in Puerto Rico

Ninety-seven people died from a fire that occurred in the Dupont Plaza Hotel in Puerto Rico on 31 Dec. 1986. All, except four who died later in the hospital, were found dead at the scene. All of the fatalities at the hotel (except for eight) were burned beyond recognition. Blood from seventy-eight of the victims was screened for carboxyhemoglobin at the Institute for Forensic Sciences in Puerto Rico and was then sent to the National Institute of Standards and Technology, Gaithersburg, Maryland, for analysis of carboxyhemoglobin and cyanide concentrations. The blood data indicated that carbon monoxide and hydrogen cyanide, singly or combined, were probably not responsible for the majority of the deaths that occurred in the badly burned victims. On the other hand, the significantly higher carboxyhemoglobin in the nonburned victims indicated that carbon monoxide alone or combined with hydrogen cyanide probably played a major role in the cause of their deaths.

Toxicological interactions between carbon monoxide and carbon dioxide

Fischer 344 male rats were subjected to 30-min individual or combined exposures of carbon monoxide (CO) and carbon dioxide (CO2). All deaths from CO occurred during the exposures, and the LC50 values were 4600 and 5000 ppm, depending on experimental conditions. Animals exposed to CO2 concentrations ranging from 1.3 to 14.7% for 30 min were neither incapacitated nor fatally injured. The addition of nonlethal concentrations of CO2 (1.7 to 17.3%) to sublethal concentrations of CO (2500 to 4000 ppm) caused deaths of the exposed rats both during and following (up to 24 h) the 30-min exposures. The most toxic combination of these two gases (2500 ppm CO plus 5% CO2) increased the rate of carboxyhemoglobin (COHb) formation 1.5 times that found in rats exposed to 2500 ppm of CO alone. The COHb equilibrium levels were the same. Exposure to both CO and CO2 produced a greater degree of acidosis and a longer recovery time than that observed with either single gas. The results fit a mathematical model indicating a synergistic interaction. Combustion of 11 materials at their LC50 values indicated that CO was probably the primary toxicant in one case and that the combined CO plus CO2 was the cause of the deaths in three other cases. Additional fire gases need to be studied to explain deaths from the other materials.

Inter- and intragenerational transmission of a human mitochondrial DNA heteroplasmy among 13 maternally-related individuals and differences between and within tissues in two family members ☆

The transmission of a C16,291C/T heteroplasmy in the HV1 region of human mitochondrial DNA (mtDNA) was examined in buccal cells from 13 maternally-related individuals across three generations and in additional tissues (hair, blood, or finger nails) from three members of this family. The ratio of C:T at nucleotide position (np) 16,291 showed wide intra- and intergenerational variation as well as tissue variation within individuals. Our results demonstrate that one or two sequence differences between samples in the mtDNA does not warrant an exclusion. To avoid false exclusions especially when comparing mtDNA from hair samples, we recommend the analysis of as many samples as possible in order to minimize the possibility that the detection of a rare polymorphism in a single sample would be considered an exclusion when it is really a match. The observation that the transmission of a mtDNA heteroplasmy from one individual to her offspring is likely to differ among the first-generation offspring and between that generation and subsequent generations lends further credence to the bottleneck theory of inheritance of human mtDNA.

New research avenues in toxicology: 7-gas N-gas model, toxicant suppressants, and genetic toxicology☆☆☆

Three research areas -- a 7-gas N-Gas Model, toxicant suppressants, and genetic toxicology -- are presented as new research approaches in toxicology. The current 6-gas N-Gas Model predicts the toxic potency of the combustion products of materials based on the toxicological interactions of the fire gases carbon monoxide (CO), carbon dioxide (CO2), low oxygen (O2) concentrations, hydrogen cyanide (HCN), hydrogen chloride, and hydrogen bromide. The present research includes nitrogen dioxide (NO2) in a new 7-gas model which incorporates the synergistic effects of NO2 and CO2, the antagonistic effects of NO2 and HCN, and the additive effects of NO2 with CO and low O2. The area of toxicant suppressants concerns chemicals, which when added to a material, will inhibit or reduce the concentration of a specific toxic gas normally generated during thermal decomposition of that material. The effectiveness of this approach was demonstrated at the US National Institute of Standards and Technology when HCN generation was reduced by 90% and the resultant toxicity of the combustion products was lowered by 50% when a flexible polyurethane (FPU) foam was treated with 0.1% (by weight) cuprous oxide (Cu2O). Although melamine-treated FPU foams are being promoted as more fire safe than standard foams, a melamine-treated foam generated 10 times more HCN than a foam without melamine. The addition of Cu2O to this melamine foam also reduced the HCN generation by 90%. The genetic toxicology research entails the examination of DNA damage that results from the exposure of human cells to various environmental toxicants and gases.

Comparison of the complete mtDNA genome sequences of human cell lines--HL-60 and GM10742A--from individuals with pro-myelocytic leukemia and leber hereditary optic neuropathy, respectively, and the inclusion of HL-60 in the NIST human mitochondrial DNA standard reference material--SRM 2392-I.

Forensic and clinical laboratories benefit from DNA standard reference materials (SRMs) that provide the quality control and assurance that their results from sequencing unknown samples are correct. Therefore, the mitochondrial DNA (mtDNA) genome of HL-60, a promyelocytic leukemia cell line, has been completely sequenced by four laboratories and will be available to the forensic and medical communities in the spring of 2003; it will be called National Institute of Standards and Technology (NIST) SRM 2392-I. NIST human mtDNA SRM 2392 will continue to be available and includes the DNA from two apparently healthy individuals. Both SRM 2392 and 2392-I contain all the information (e.g. the sequences of 58 unique primer sets) needed to use these SRMs as positive controls for the amplification and sequencing any DNA. Compared to the templates in SRM 2392, the HL-60 mtDNA in SRM 2392-I has two tRNA differences and more polymorphisms resulting in amino acid changes. Four of these HL-60 mtDNA polymorphisms have been associated with Leber Hereditary Optic Neuropathy (LHON), one as an intermediate mutation and three as secondary mutations. The mtDNA from a cell line (GM10742A) from an individual with LHON was also completely sequenced for comparison and contained some of the same LHON mutations. The combination of these particular LHON associated mutations is also found in phylogenetic haplogroup J and its subset, J2, and may only be indicative that HL-60 belongs to haplogroup J, one of nine haplogroups that characterize Caucasian individuals of European descent or may mean that haplogroup J is more prone to LHON. Both these mtDNA SRMs will provide enhanced quality control in forensic identification, medical diagnosis, and single nucleotide polymorphism detection.

MitoAnalyzer, a computer program and interactive web site to determine the effects of single nucleotide polymorphisms and mutations in human mitochondrial DNA ☆

MitoAnalyzer provides information about the effects of single nucleotide polymorphisms (SNPs) and mutations in human mitochondrial DNA (mtDNA). This program determines if a single base pair (bp) change is in the non-coding or coding region, in the first, second or third bp of the codon, in a rRNA, tRNA or a protein, causes an amino acid (aa) change, the nature of that change, the position of the aa change in the protein, and the new aa sequence of the changed protein. Mutations associated with published mitochondrial diseases are noted. This program, thus, facilitates rapid analysis and evaluation of SNPs and mutations in human mtDNA.