Glenn D. Ritchie

BIOLOGICAL AND HEALTH EFFECTS OF EXPOSURE TO KEROSENE-BASED JET FUELS AND PERFORMANCE ADDITIVES

Over 2 million military and civilian personnel per year (over 1 million in the United States) are occupationally exposed, respectively, to jet propulsion fuel-8 (JP-8), JP-8 +100 or JP-5, or to the civil aviation equivalents Jet A or Jet A-1. Approximately 60 billion gallons of these kerosene-based jet fuels are annually consumed worldwide (26 billion gallons in the United States), including over 5 billion gallons of JP-8 by the militaries of the United States and other NATO countries. JP-8, for example, represents the largest single chemical exposure in the U.S. military (2.53 billion gallons in 2000), while Jet A and A-1 are among the most common sources of nonmilitary occupational chemical exposure. Although more recent figures were not available, approximately 4.06 billion gallons of kerosene per se were consumed in the United States in 1990 (IARC, 1992). These exposures may occur repeatedly to raw fuel, vapor phase, aerosol phase, or fuel combustion exhaust by dermal absorption, pulmonary inhalation, or oral ingestion routes. Additionally, the public may be repeatedly exposed to lower levels of jet fuel vapor/aerosol or to fuel combustion products through atmospheric contamination, or to raw fuel constituents by contact with contaminated groundwater or soil. Kerosene-based hydrocarbon fuels are complex mixtures of up to 260+ aliphatic and aromatic hydrocarbon compounds (C 6 -C 17+ ; possibly 2000+ isomeric forms), including varying concentrations of potential toxicants such as benzene, n-hexane, toluene, xylenes, trimethylpentane, methoxyethanol, naphthalenes (including polycyclic aromatic hydrocarbons [PAHs], and certain other C 9 -C 12 fractions (i.e., n-propylbenzene, trimethylbenzene isomers). While hydrocarbon fuel exposures occur typically at concentrations below current permissible exposure limits (PELs) for the parent fuel or its constituent chemicals, it is unknown whether additive or synergistic interactions among hydrocarbon constituents, up to six performance additives, and other environmental exposure factors may result in unpredicted toxicity. While there is little epidemiological evidence for fuel-induced death, cancer, or other serious organic disease in fuel-exposed workers, large numbers of self-reported health complaints in this cohort appear to justify study of more subtle health consequences. A number of recently published studies reported acute or persisting biological or health effects from acute, subchronic, or chronic exposure of humans or animals to kerosene-based hydrocarbon fuels, toconstituent chemicals of these fuels, or to fuel combustion products. This review provides an in-depth summary of human, animal, and in vitro studies of biological or health effects from exposure to JP-8, JP-8 +100, JP-5, Jet A, Jet A-1, or kerosene.

A REVIEW OF THE NEUROTOXICITY RISK OF SELECTED HYDROCARBON FUELS

Over 1.3 million civilian and military personnel are occupationally exposed to hydrocarbon fuels, emphasizing gasoline, jet fuel, diesel fuel, or kerosene. These exposures may occur acutely or chronically to raw fuel, vapor, aerosol, or fuel combustion exhaust by dermal, respiratory inhalation, or oral ingestion routes, and commonly occur concurrently with exposure to other chemicals and stressors. Hydrocarbon fuels are complex mixtures of 150-260+ aliphatic and aromatic hydrocarbon compounds containing varying concentrations of potential neurotoxicants including benzene, n-hexane, toluene, xylenes, naphthalene, and certain n-C9-C12 fractions (n-propylbenzene, trimethylbenzene isomers). Due to their natural petroleum base, the chemical composition of different hydrocarbon fuels is not defined, and the fuels are classified according to broad performance criteria such as flash and boiling points, complicating toxicological comparisons. While hydrocarbon fuel exposures occur typically at concentrations below permissible exposure limits for their constituent chemicals, it is unknown whether additive or synergistic interactions may result in unpredicted neurotoxicity. The inclusion of up to six performance additives in existing fuel formulations presents additional neurotoxicity challenge. Additionally, exposures to hydrocarbon fuels, typically with minimal respiratory or dermal protection, range from weekly fueling of personal automobiles to waist-deep immersion of personnel in raw fuel during maintenance of aircraft fuel tanks. Occupational exposures may occur on a near daily basis for from several months to over 20 yr. A number of published studies have reported acute or persisting neurotoxic effects from acute, subchronic, or chronic exposure of humans or animals to hydrocarbon fuels, or to certain constituent chemicals of these fuels. This review summarizes human and animal studies of hydrocarbon fuel-induced neurotoxicity and neurobehavioral consequences. It is hoped that this review will support ongoing attempts to review and possibly revise exposure standards for hydrocarbon fuels.Over 1.3 million civilian and military personnel are occupationally exposed to hydrocarbon fuels, emphasizing gasoline, jet fuel, diesel fuel, or kerosene. These exposures may occur acutely or chronically to raw fuel, vapor, aerosol, or fuel combustion exhaust by dermal, respiratory inhalation, or oral ingestion routes, and commonly occur concurrently with exposure to other chemicals and stressors. Hydrocarbon fuels are complex mixtures of 150-260+ aliphatic and aromatic hydrocarbon compounds containing varying concentrations of potential neurotoxicants including benzene, n-hexane, toluene, xylenes, naphthalene, and certain n-C9-C12 fractions (n-propylbenzene, trimethylbenzene isomers). Due to their natural petroleum base, the chemical composition of different hydrocarbon fuels is not defined, and the fuels are classified according to broad performance criteria such as flash and boiling points, complicating toxicological comparisons. While hydrocarbon fuel exposures occur typically at concentrations below permissible exposure limits for their constituent chemicals, it is unknown whether additive or synergistic interactions may result in unpredicted neurotoxicity. The inclusion of up to six performance additives in existing fuel formulations presents additional neurotoxicity challenge. Additionally, exposures to hydrocarbon fuels, typically with minimal respiratory or dermal protection, range from weekly fueling of personal automobiles to waist-deep immersion of personnel in raw fuel during maintenance of aircraft fuel tanks. Occupational exposures may occur on a near daily basis for from several months to over 20 yr. A number of published studies have reported acute or persisting neurotoxic effects from acute, subchronic, or chronic exposure of humans or animals to hydrocarbon fuels, or to certain constituent chemicals of these fuels. This review summarizes human and animal studies of hydrocarbon fuel-induced neurotoxicity and neurobehavioral consequences. It is hoped that this review will support ongoing attempts to review and possibly revise exposure standards for hydrocarbon fuels.

A review of the effects of uranium and depleted uranium exposure on reproduction and fetal development.

Depleted uranium (DU) is used in armor-penetrating munitions, military vehicle armor, and aircraft, ship and missile counterweighting/ballasting, as well as in a number of other military and commercial applications. Recent combat applications of DU alloy [i.e., Persian Gulf War (PGW) and Kosovo peacekeeping objective] resulted in human acute exposure to DU dust, vapor or aerosol, as well as chronic exposure from tissue embedding of DU shrapnel fragments. DU alloy is 99.8% 238Uranium, and emits approximately 60% of the alpha, beta, and gamma radiation found in natural uranium (4.05×10-7 Ci/g DU alloy). DU is a heavy metal that is 160% more dense than lead and can remain within the body for many years and slowly solubilize. High levels of urinary uranium have been measured in PGW veterans 10 years after exposure to DU fragments and vapors. In rats, there is strong evidence of DU accumulation in tissues including testes, bone, kidneys, and brain. In vitro tests indicate that DU alloy may be both genotoxic and mutagenic, whereas a recent in vivo study suggests that tissue-embedded DU alloy may be carcinogenic in rats. There is limited available data for reproductive and teratological deficits from exposure to uranium per se, typically from oral, respiratory, or dermal exposure routes. Alternatively, there is no data available on the reproductive effects of DU embedded. This paper reviews published studies of reproductive toxicity in humans and animals from uranium or DU exposure, and discusses ongoing animal research to evaluate reproductive effects in male and female rats embedded with DU fragments, and possible consequences in F1 and F2 generations.

Toxicity of chemical mixtures:Proteomic analysis of persisting liver and kidney protein alterations induced by repeated exposure of rats to JP‐8 jet fuel vapor

Male Sprague‐Dawley rate were exposed by whole body inhalation to 1000 mg/m3 ± 10% JP‐8 jet fuel vapor or room air control conditions for 6 h/day, 5 days/week for six consecutive weeks. Following a rest period of 82 days rats were sacrificed, and liver and kidney tissues examined by proteomic methods for both total protein abundance and protein charge modification. Kidney and lung samples were solubilized and separated via large scale, high resolution two‐dimensional electrophoresis (2‐DE) and gel patterns scanned, digitized and processed for statistical analysis. Through the use of peptide mass fingerprinting, confirmed by sequence tag analysis, three altered proteins were identified and quantified. Numerical, but not significantly different increases were found in total abundance of lamin A (NCBI Accession No. 1346413) in the liver, and of 10‐formyltetrahydrofolate dehydrogenase (10‐FTHF DH, #1346044) and glutathione‐S‐transferase (GST; #2393724) in the kidneys of vapor‐exposed subjects. Protein charge modification index (CMI) analysis indicated significant alterations (P < 0.001) in expressed lamin A and 10‐FTHF DH. These persisting changes in liver and kidney proteins are discussed in terms of possible alterations in the functional capacity of exposed subjects.

EFFECTS OF REPEATED EXPOSURE OF RATS TO JP-5 OR JP-8 JET FUEL VAPOR ON NEUROBEHAVIORAL CAPACITY AND NEUROTRANSMITTER LEVELS

The U.S. Naval Service is anticipating transition from the nearly exclusive use of JP-5 jet fuel to predominant use of JP-8, consistent with the primary utilization by the U.S. Army, U.S. Air Force, and the militaries of most NATO countries. To compare the relative risk of repeated exposure to JP-5 versus JP-8 vapor, groups of 32 male Sprague-Dawley rats each were exposed for 6 h/d, 5 d/wk for 6 wk (180 h) to JP-8 jet fuel vapor (1,000 +/- 10% mg/m3), IP-5 vapor (1,200 +/- 10% mg/m3), or room air control conditions. Following a 65-d rest period, rats completed 10 tests selected from the Neurobehavioral Toxicity Assessment Battery (NTAB) to evaluate changes in performance capacity. Repeated exposure to JP-5 resulted in significant effects on only one test, forelimb grip strength (FGS), while exposure to JP-8 vapor resulted in a significant difference versus controls on appetitive reinforcer approach sensitization (ARAS). Rats were further evaluated for concentrations of major neurotransmitters and metabolites in five brain regions and in the blood serum. Levels of dopamine, the dopamine metabolite dihydroxyphenylacetic acid (DOPAC), and the serotonin metabolite homovanillic acid (HVA) were significantly modulated in various brain regions, as measured 85+ d postexposure. Similarly, serum levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) were differentially modulated following JP-8 or JP-5 exposure. Results are compared to previously published research evaluating the neurotoxicity of repeated exposure to other hydrocarbon fuels and solvents.

ACUTE EFFECTS OF A BICYCLOPHOSPHATE NEUROCONVULSANT ON MONOAMINE NEUROTRANSMITTER AND METABOLITE LEVELS IN THE RAT BRAIN

Naive male Sprague-Dawley rats were injected intraperitoneally (i.p.) with the bicyclophosphate convulsant trimethylolpropane phosphate (TMPP) at dose levels from 0.2 to 0.6 mg/kg. Rats were observed for convulsive activity, and were sacrificed 15 min posttreatment. Levels of the monoamine neurotransmitters norepinephrine (NE), epinephrine (EPI), dopamine (DA), and serotonin (5-HT) and the major metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) were assayed in forebrain, midbrain, hindbrain, cerebellum and brainstem regions. Neurotransmitter and metabolite levels were compared between control rats and rats that did and did not experience seizures. TMPP administration induced significant decreases in levels of measured neurotransmitters that varied as a function of brain region, dose, and expression of the seizure activity. These results show that tonic or tonic-clonic seizures induced by TMPP administration (0.6 mg/kg) are reliably associated with regional decreases in serotonin, dopamine, and norepinephrine. Convulsive activity resulting from lower dose administrations (0.2-0.4 mg/kg) of TMPP result only in decreased regional levels of serotonin.Naive male Sprague-Dawley rats were injected intraperitoneally (ip) with the bicyclophosphate convulsant trimethylolpropane phosphate (TMPP) at dose levels from 0.2 to 0.6 mg/kg. Rats were observed for convulsive activity, and were sacrificed 15 min posttreatment. Levels of the monoamine neurotransmitters norepinephrine (NE), epinephrine (EPI), dopamine (DA) , and serotonin (5-HT) and the major metabolites 3,4- dihydroxyphenylacetic acid ( DOPAC) , homovanillic acid ( HVA) , and 5-hydroxyindoleacetic acid ( 5-HIAA) were assayed in forebrain, midbrain, hindbrain, cerebellum and brainstem regions. Neurotransmitter and metabolite levels were compared between control rats and rats that did and did not experience seizures. TMPP administration induced significant decreases in levels of measured neurotransmitters that varied as a function of brain region, dose, and expression of the seizure activity. These results show that tonic or tonic-clonic seizures induced by TMPP administration (0.6 mg/ kg) are reliably a...

Gene expression profiles in the rat central nervous system induced by JP-8 jet fuel vapor exposure

Jet propulsion fuel-8 (JP-8) is the predominant fuel for military land vehicles and aircraft used in the US and NATO. Occupational exposure to jet fuel in military personnel has raised concern for the health risk associated with such exposure in the Department of Defense. Clinical studies of humans chronically exposed to jet fuel have suggested both neurotoxicity and neurobehavioral deficits. We utilized rat neurobiology U34 array to measure gene expression changes in whole brain tissue of rats exposed repeatedly to JP-8, under conditions that simulated possible occupational exposure (6 h/day for 91 days) to JP-8 vapor at 250, 500, and 1000 mg/m(3), respectively. Our studies revealed that the gene expression changes of exposure groups can be divided into two main categories according to their functions: (1). neurotransmitter signaling pathways; and (2). stress response. The implications of these gene expression changes are discussed.

APPLICATION OF NEUROBEHAVIORAL TOXICOLOGY METHODS TO THE MILITARY DEPLOYMENT TOXICOLOGY ASSESSMENT PROGRAM

The military Tri-Service (Army, Navy & Marines, Air Force) Deployment Toxicology Assessment Program (DTAP) represents a 30-year (1996–2026) planning effort to implement comprehensive systems for the protection of internationally deployed troops against toxicant exposures. A major objective of DTAP is the implementation of a global surveillance system to identify chemicals with the potential to reduce human performance capacity. Implementation requires prior development of complex human risk assessment models, known collectively as the Neurobehavioral Toxicity Evaluation Instrument (NTEI), based on mathematical interpolation of results from tissue-based and in vivo animal studies validated by human performance assessment research. The Neurobehavioral Toxicity Assessment Group (NTAG) at the Naval Health Research Center Detachment-Toxicology (NHRC-TD), Dayton, OH, and associated academic institutions are developing and cross-validating cellular-level (NTAS), laboratory small animal (NTAB), nonhuman primate (GASP), and human-based (GASH) toxicity assessment batteries. These batteries will be utilized to develop and evaluate mathematical predictors of human neurobehavioral toxicity, as a function of laboratory performance deficits predicted by quantitative structural analysis relationship (QSAR-like) properties of potential toxicants identified by international surveillance systems. Finally, physiologically-based pharmacokinetic (PBPK) and pharmacodynamic (PBPD) modeling of NTAS, NTAB, GASP, GASH data will support multi-organizational development and validation of the NTEI. The validated NTEI tool will represent a complex database management system, integrating global satellite surveillance input to provide real-time decision-making support for deployed military personnel.

An overview of the development, validation, and application of neurobehavioral and neuromolecular toxicity assessment batteries: potential applications to combustion toxicology

Currently, there are few alternatives to the use of animals in toxicology for human risk assessment. Neurobehavioral toxicology is an emerging area in which complex performance capacity is evaluated during or following toxicological exposure. While a number of single tests and a few more complex neurobehavioral batteries exist, no fully validated and comprehensive neurobehavioral toxicity assessment battery has yet been developed. The Neurobehavioral Toxicity Assessment Battery (NTAB) is a multi-test battery being developed by the Naval Medical Research Institute Detachment (Toxicology) (NMRI/TD) to categorize the potential neurobehavioral toxicity of compounds of Navy interest, especially those found in combustion atmospheres. The NTAB is intended to identify specific areas of deficit (e.g. motivational, sensory, motor, and cognitive) from complex changes in performance induced by toxic exposures, as well as to provide a mechanism to evaluate recovery of neurobehavioral integrity. Portions of the NTAB have been successfully used to assess the risk of brief exposure to low concentrations of combustion gases, including smoke from electrical aircraft fires, ozone-depleting substances and their replacements, and the novel neuroconvulsant trimethylolpropane phosphate. The goal of the NMRI/TD Neurobehavioral Toxicology Group and the Tri-Service Toxicology Consortiums neurobehavioral toxicology program is the incorporation of more molecular techniques involving neurophysiology, neuropharmacology, in vivo electrochemistry, and real-time microdialysis for correlative use with the neurobehavioral battery in human risk assessment. This overview discusses the application of neurobehavioral and neuromolecular endpoint test batteries to combustion toxicology.

Repeated exposure to trimethylolpropane phosphate induces central nervous system sensitization and facilitates electrical kindling.

Trimethylolpropane phosphate (TMPP), pentylenetetrazol (PTZ) and N-methyl-beta-carboline-3-carboxamide (FG-7142) were evaluated and compared for facilitation of electrical kindling in freely moving rats. Stimulating/recording electrodes were implanted in the left amygdala (LAD), right amygdala (RAD) and left bed nucleus (LBN) of the stria terminalis. TMPP (0.275 mg/kg), PTZ (20 mg/kg), FG-7142 (7.5 mg/kg) or vehicle was administered intraperitoneally (i.p.) to separate groups of rats 3 times/week for 10 weeks. Stimulation of the LAD (0.1 Hz, 0.1-ms duration, 280-1500 microA, 20 pulses) 24 h following the drug administration evoked epileptiform after-discharges (ADs) in the LBN and RAD of 12.5% and 17% of rats after the seventh dose of TMPP and PTZ, respectively, and in 20% of rats from the LBN and RAD after the ninth and nineteenth dose of FG-7142, respectively. The same stimulation also induced myoclonic jerks after nine doses of TMPP or PTZ, or after thirteen doses of FG-7142 in 25%, 30% and 20% of animals tested, respectively. Chemically kindled clonic seizures were observed in 100% of TMPP or FG-7142 and 50% of PTZ treated rats by the thirtieth dosing. Control animals exhibited neither behavioral nor electrographic seizures to vehicle injection or to the LAD stimulation. Kindling stimulation applied to the LAD (60 Hz, 2-s train duration, 20-1500 microA, 0.1-ms pulse duration) 4 weeks following the completion of drug treatments evoked epileptic after-discharges from the LAD, LBN and RAD in all treated groups, with generally decreased threshold and latency to onset of after-discharges, compared to vehicle controls. The present study suggests that repeated exposure of rats to sub-convulsive doses of TMPP, PTZ and FG-7142 induces long-term central nervous system sensitization that may be related to both chemical kindling and the facilitation of electrical kindling.