Andreas F. Lehner

Diagnostic Analysis of Veterinary Dried Blood Spots for Toxic Heavy Metals Exposure

Dried blood spots (DBS) on filter paper have been used in human medicine since the 1960s, predominantly for screening in-borne metabolic disorders and more recently, for toxicology. Despite its 50-year existence, this technology has not been adopted by veterinarians for routine diagnoses and research. We have validated a novel DBS analytical procedure for the routine measurement of toxic heavy metals using 50 µL of whole blood on a single DBS by inductively coupled plasma mass spectrometry (ICP-MS). Targeted heavy metals are arsenic, cadmium, mercury, lead, selenium and thallium. The limits of quantitation (LOQ) on DBS are: arsenic 1.7 µg/L, cadmium 4.0 µg/L, mercury 13.7 µg/L, lead 13.3 µg/L, selenium 6.3 µg/L and thallium 1.5 µg/L. These LOQs suffice for routine diagnoses of heavy metal intoxication in domesticated and wildlife species as well as for basic, applied and epidemiological studies. The technique is ideal for population studies involving investigations of wildlife exposure to heavy metals and other environmental pollutants. The small blood volume involved (50 µL) makes it feasible to study small animals (birds, reptiles, amphibians and small mammals) that were previously excluded, or difficult to study due to the relatively large sample volumes required by current gold standard blood collection techniques.

Examination of Eurasian Griffon Vultures (Gyps fulvus fulvus) in Israel for Exposure to Environmental Toxicants Using Dried Blood Spots

The griffon vulture (Gyps fulvus) is one of seven species of Old World Gyps vultures found over a wide range from the Iberian peninsula in the west through the Balkans, Turkey, and the Middle East to India in the east. The population of the griffon vultures in Israel has suffered a dramatic decrease, and in recent years productivity has been severely reduced. In this study, whole-blood samples taken from 25 apparently healthy griffon vultures at various stages of maturity were examined to investigate whether the vultures are being excessively exposed to environmental contaminants that might deleteriously affect their reproduction. Five groups of environmental contaminants, comprising toxic elements, organochlorine pesticides, polychlorinated biphenyls, polybrominated diphenyl ethers, and perfluorinated compounds, were monitored in dried blood spots. Results of the analyses showed low levels of exposure of griffon vultures to environmental contaminants compared with the sparse data available on griffon vultures and other diurnal raptors in other countries.

Organophosphate Ester Flame Retardant-Induced Acute Intoxications in Dogs

IntroductionFlame retardants have wide industrial applications and are incorporated into articles found in automobiles and home environments, including seat cushions. These compounds differ widely chemically and in their toxic potential. We report here two cases involving dogs following ingestion of car seat cushions impregnated with organophosphate ester fire retardants.Case ReportsTwo case reports are presented. Two adult American Pit Bull dogs were presented at an emergency clinic with acute signs of central nervous system excitation including seizures. The most severely affected dog died 15xa0min after presentation, while the less affected dog fully recovered following treatment. In the second case, both a German Shepherd and a Rottweiler were found dead in the morning after they were left in a car overnight. A comprehensive toxicological analysis of samples from both cases revealed the presence of significant amounts (>2xa0ppm) of tris(2-chloroethyl)phosphate (TCEP) in stomach contents. This compound is a known inducer of epileptic seizures. Some other structurally related organophosphate ester compounds were found, and their role in the acute intoxications reported here is not known and remains to be determined.ConclusionThis is the first report linking acute deaths in dogs to the ingestion of car seat cushions found to contain large amounts of TCEP, an organophosphate ester compound. It is highly likely that this compound caused death through its known seizure-inducing activity.

Chronic lead exposure is epidemic in obligate scavenger populations in eastern North America

Lead is a prominent and highly toxic contaminant with important impacts to wildlife. To understand the degree to which wildlife populations are chronically exposed, we quantified lead levels within American black vultures (Coragyps atratus; BLVU) and turkey vultures (Cathartes aura; TUVU), two species that are useful as environmental sentinels in eastern North America. Every individual sampled (n=108) had bone lead levels indicative of chronic exposure to anthropogenic lead (BLVU: x¯=36.99 ± 55.21 mg Pb/kg tissue (±SD); TUVU: x¯=23.02 ± 18.77 mg/kg). Only a few showed evidence of recent lead exposure (BLVU liver: x¯=0.78 ± 0.93 mg/kg; TUVU liver: x¯=0.55 ± 0.34 mg/kg). Isotopic ratios suggested multiple potential sources of lead including ammunition, gasoline, coal-fired power plants, and zinc smelting. Black and turkey vultures range across eastern North America, from Quebec to Florida and individuals may traverse thousands of kilometers annually. The extent to which vultures are exposed suggests that anthropogenic lead permeates eastern North American ecosystems to a previously unrecognized degree. Discovery of an epidemic of chronic lead exposure in such widespread and common species and the failure of soft-tissue sampling to diagnose this pattern has dramatic implications for understanding modern wildlife and human health concerns.

Liquid chromatographic-electrospray mass spectrometric determination of 1-methyl-4-phenylpyridine (MPP+) in discrete regions of murine brain.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is widely used as a neurotoxin in several models of Parkinson’s disease in mice. MPTP is metabolized to 1-methyl-4-phenylpyridinium (MPP+), which is a mitochondrial toxicant of central dopamine (DA) neurons. There are species, strain, and age differences in sensitivity to MPTP. Simultaneous measurement of the MPTP active metabolite MPP+ and dopamine (DA) in the brain would be helpful in mechanistic studies of this neurotoxin. The objective of this study was to develop a liquid chromatography–mass spectrometry (LC/MS) method for analysis of MPTP and MPP+ in brain tissue and correlate these in the same sample with changes in DA measured via HPLC coupled with electrochemical detection. Twenty-five C57BL/6J7 8-week old female mice were used in the study. Mice were given a single subcutaneous injection of MPTP (20u2009mg/kg) and were sacrificed 1, 2, 4, or 8u2009h later. Zero time control mice received an injection of 0.9% normal saline (10u2009ml/kg) and were killed 1u2009h later. Brains were rapidly harvested and quickly frozen, and microdissected brain regions were placed in 0.1u2009M phosphate-citric acid buffer containing 20% methanol (pH 2.5). A new LC/MS method was successfully developed that utilized selected reaction monitoring (SRM) of MPP+ m/z 170→127, 170→128, and 170→154 fragmentation for quantitation and area ratios (m/z 127)/(m/z 128) and (m/z 154)/(128) for identity confirmation. A similar SRM strategy from m/z 174 was unable to detect any significant levels of MPTP down to 0.4 ppb. According to this method, MPP+ was detected in the nucleus accumbens (NA) and the striatum (ST), with the levels in the NA being 3-times higher than those in the ST. The advantage of this approach is that the tissue buffer used in this procedure allowed concurrent measurement of striatal DA, thus enabling direct correlation between accumulation of tissue MPP+ and depletion of DA concentrations in discrete regions of the brain.

A Cytochrome P450–Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes

Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0–14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

Structure, function and carcinogenicity of metabolites of methylated and non-methylated polycyclic aromatic hydrocarbons: a comprehensive review.

Abstract The Unified Theory of PAH Carcinogenicity accommodates the activities of methylated and non-methylated polycyclic aromatic hydrocarbons (PAHs) and states that substitution of methyl groups on meso-methyl substituted PAHs with hydroxy, acetoxy, chloride, bromide or sulfuric acid ester groups imparts potent cancer producing properties. It incorporates specific predictions from past researchers on the mechanism of carcinogenesis by methyl-substituted hydrocarbons, including (1) requirement for metabolism to an ArCH2X type structure where X is a good leaving group and (2) biological substitution of a meso-methyl group at the most reactive center in non-methylated hydrocarbons. The Theory incorporates strong inferences of Fieser: (1) The mechanism of carcinogenesis involves a specific metabolic substitution of a hydrocarbon at its most reactive center and (2) Metabolic elimination of a carcinogen is a detoxifying process competitive with that of carcinogenesis and occurring by a different mechanism. According to this outlook, chemical or biochemical substitution of a methyl group at the reactive meso-position of non-methylated hydrocarbons is the first step in the mechanism of carcinogenesis for most, if not all, PAHs and the most potent metabolites of PAHs are to be found among the meso methyl-substituted hydrocarbons. Some PAHs and their known or potential metabolites and closely related compounds have been tested in rats for production of sarcomas at the site of subcutaneous injection and the results strongly support the specific predictions of the Unified Theory.

River otters as biomonitors for organochlorine pesticides, PCBs, and PBDEs in Illinois

The North American river otter (Lontra canadensis) is a biomonitor for organohalogenated compounds (OHCs) associated with a wide range of deleterious health effects in wildlife and humans. We determined concentrations of twenty OHCs in livers of 23 river otters salvaged by the Illinois Department of Natural Resources from 2009 to 2011, determined sex-dependent distribution of OHCs, and compared our results to the reported concentrations of four OHCs in Illinois river otters from 1984 to 1989. Since these contaminants have been banned for over 30 years, we predicted smaller mean concentrations than those previously reported in Illinois otters. We detected eleven of twenty OHCs; PCBs (polychlorinated biphenyls), dieldrin, and 4,4-DDE (dichlorodiphenyldichloroethylene) were present in the greatest mean concentrations. We report the largest mean concentration of dieldrin to date in the liver of North American river otters (mean: 174, range: 14.4-534 parts per billion wet wt [ppb]). Mean PCB concentrations were significantly higher in males (mean: 851; range: 30-3450 ppb) than females (mean: 282; range: 40-850 ppb; p=0.04). Mean concentrations of dieldrin were greater than those detected in otters from 1984 to 1989 (mean: 90; range: 30-130 ppb; p<0.05). Our results suggest OHC exposure remains a concern. Future research in Illinois should focus on evaluating OHCs exposures, particularly dieldrin, at the watershed level.

Determination of Organochlorine Pesticides in Wildlife Liver and Serum Using Gas Chromatography Tandem Quadrupole Mass Spectrometry

A single laboratory validation of a quantitative capillary gas chromatography tandem quadrupole mass spectrometer (GC-MS/MS) method utilizing a Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) approach for the extraction of 24 organochlorine analytes in liver and blood serum is presented here. The QuEChERS approach utilizes an acetonitrile extraction, partitioning facilitated by the addition of salts and a dispersive solid phase extraction cleanup. This method simultaneously monitored 24 organochlorine pesticide residues representing four different classes, including chlordecone, cyclodienes, dichlorodiphenylethanes, and hexachlorocyclohexanes. Calculated limits of detection (LOD) varied from 0.002 to 2.4 ppb and limits of quantification (LOQ) varied from 0.01 to 7.4 ppb. This multi-residue method proved to be a sensitive approach to the measurement of persistent organic pollutants in biological matrices.

Essential, trace and toxic element concentrations in the liver of the world's largest bony fish, the ocean sunfish (Mola mola).

No studies document essential (calcium, magnesium, phosphorus, potassium, sodium), trace (barium, boron, chromium, cobalt, copper, iron, manganese, molybdenum, selenium, zinc) or toxic element (antimony, arsenic, cadmium, lead, mercury, thallium) concentrations in any members of the family Molidae, including the worlds largest bony fish, the ocean sunfish (Mola mola). Here, we analyzed 21 elements in the liver of one M. mola. These values were compared to liver concentrations in multiple species with spatial and dietary overlap. Concentrations of calcium (3339 ppm wet weight) and iron (2311 ppm wet weight) were extremely elevated in comparison to a number of other fish species, indicating that calcium and/or iron toxicity may have occurred in this animal. Concentrations of toxic elements were generally low, with the exception of cadmium (3.5 ppm). This study represents the first report of essential, trace and toxic elements in this species.