Evaluation of the hazards of industrial exposure to tricresyl phosphate: a review and interpretation of the literature.

Commercial tricresyl phosphate (TCP) is a heterogeneous mixture of isomers and aryl phosphate congeners, known for many years to induce delayed neurotoxicity (OPIDN) in humans and experimental animals. In the past the isomer tri-o-cresyl phosphate (TOCP) was thought to be the component primarily responsible for OPIDN. It is now clear that other constituents, particularly the mono-o-esters, are not only neurotoxic but also may be more potent than pure TOCP. As a generality, the toxicity potential of a particular brand of TCP is related to its content of o-phenolic residues, whereby the maximal potential is reached when o-phenolics are 33% of the mix. Historically, human TCP toxicity has resulted from inadvertent or intentional contamination of foodstuffs or beverages. TCP products with high ortho-residues synthesized by older manufacturing methods were involved in most of these cases, and were likely much more neurotoxic milligram for milligram than TOCP. Because of the great variability of TCP products, there are no conventional workplace exposure standards. Based upon data from the hen and cat, estimated human safe exposure rates for pure TOCP are estimated to be 2.5 mg/kg for a single dose, and 0.13 mg/kg/d for repetitive exposures. These levels may also be applied to TCP when o-methyl-phenyl, o-ethyl-phenyl, and o-xylenyl components are appropriately limited during manufacture such that the TCP product is less neurotoxic than TOCP. There have been relatively few reports of toxicity associated with the manufacture or use of TCP in commerce and industry. Low vapor pressures of the constituents preclude the presence of significant quantities of TCP vapor in the atmosphere. A lubricant or other formulation containing TCP may appear in the air as a mist. By these criteria the U.S. Petroleum Oil Mist exposure standard is protective when the formulation contains 4% or less of low ortho-TCP. Exposure calculations indicate that estimated safe levels are not likely to be exceeded in the well-regulated workplace. If it is of short duration, even a heavy fog of oil particulate may not exceed the 8-h-average inhalation exposure standard. Modern manufacturing practices tend to minimize the ortho content and thus the toxicity of TCP. Because individual TCP brands and synthesis methods vary, manufacturers should be consulted concerning the properties of their individual products.

Comparison of neurotoxic effects and potential risks from oral administration or ingestion of tricresyl phosphate and jet engine oil containing tricresyl phosphate.

Neurotoxicity of tricresyl phosphates (TCPs) and jet engine oil (JEO) containing TCPs were evaluated in studies conducted in both rat and hen. Results for currently produced samples ("conventional" and "low-toxicity") were compared with published findings on older samples to identify compositional changes and relate those changes to neurotoxic potential. Finally, a human risk assessment for exposure by oral ingestion of currently produced TCPs in JEO at 3% (JEO + 3%) was conducted. TCPs and certain other triaryl phosphates administered as single doses inhibited brain neuropathy target esterase (B-NTE; neurotoxic esterase) in the rat and the hen (hen 3.25 times as sensitive), and both species were deemed acceptable for initial screening purposes. Neither rat nor hen was sensitive enough to detect statistically significant inhibition of B-NTE after single doses of IEO + 3% "conventional" TCP. Subacute administration of 2 g/kg/d of JEO + 3% "conventional" TCP to the hen produced B-NTE inhibition (32%), which did not result in organophosphorus-induced delayed neurotoxicity (OPIDN). Subchronic administration of JEO + 3% TCP but not JEO + 1% TCP at 2 g/kg/d produced OPIDN. Thus, the threshold for OPIDN was between 20 and 60 mg "conventional" TCP/kg/d in JEO for 10 wk. The current "conventional" TCPs used in JEO and new "low-toxicity" TCPs now used in some JEO are synthesized from phenolic mixtures having reduced levels of ortho-cresol and ortho-xylenols resulting in TCPs of very high content of meta- and para-substituted phenyl moieties; this change in composition results in lower toxicity. The "conventional" TCPs still retain enough inhibitory activity to produce OPIDN, largely because of the presence of ortho-xylyl moieties; the "low-toxicity" TCPs are largely devoid of ortho substituents and have extremely low potential to cause OPIDN. The TCPs produced in the 1940s and 1950s were more than 400 times as toxic as the "low-toxicity" TCPs produced today. Analysis of the doses required to produce OPIDN in a subchronic hen study suggests that the minimum toxic dose of "conventional" TCP for producing OPIDN in a 70-kg person would be 280 mg/d, and for JEO containing 3% TCP, 9.4 g/d. Food products could be inadvertently contaminated with neat "conventional" TCP but it is unlikely that food such as cooking oil would be contaminated with enough JEO + 3% TCP to cause toxicity. Further, at the dosage required for neurotoxicity, it would be virtually impossible for a person to receive enough JEO + 3% TCP in the normal workplace (or in an aircraft) to cause such toxicity. There is no record of a JEO formulated with the modern "conventional" TCP causing human neurotoxicity.

A regulatory element in intron 1 of the cystic fibrosis transmembrane conductance regulator gene.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene exhibits a tightly regulated pattern of expression in human epithelial cells. The mechanism of this regulation is complex and is likely to involve a number of genetic elements that effect temporal and spatial expression. To date none of the elements that have been identified in the CFTR promoter regulate tissue-specific expression. We have identified a putative regulatory element within the first intron of the CFTR gene at 181+10kb. The region containing this element was first identified as a DNase I hypersensitive site that was present in cells that express the CFTR gene but absent from cells not transcribing CFTR. In vitro analysis of binding of proteins to this region of DNA sequence by gel mobility shift assays and DNase I footprinting revealed that some proteins that are only present in CFTR-expressing cells bound to specific elements, and other proteins that bound to adjacent elements were present in all epithelial cells irrespective of their CFTR expression status. When assayed in transient expression systems in a cell line expressing CFTR endogenously, this DNA sequence augmented reporter gene expression through activation of the CFTR promoter but had no effect in nonexpressing cells.

Frequency of arylsulphatase A pseudodeficiency associated mutations in a healthy population.

Arylsulphatase A (ASA, EC 3.1.6.1) is a lysosomal enzyme that catalyses cerebroside sulphate degradation. ASA deficiency is associated with metachromatic leucodystrophy (MLD), a rare autosomal recessive disorder, which is characterised by the storage of cerebroside sulphate. Low ASA activities can be also observed in clinically healthy persons, a condition termed ASA pseudodeficiency. Two mutations responsible for the majority of pseudodeficiency alleles have been defined in the ASA gene. These are both A-->G transitions. One causes an asparagine to serine substitution (N350S). The second changes the first polyadenylation signal downstream of the stop codon (1524 + 95A-->G), which causes a severe deficiency of one ASA mRNA species. The incidence of the pseudodeficiency allele is estimated to be high in the general population and can be found in families carrying MLD associated mutations. We report a reliable stratagem for detecting the two PD associated mutations separately, which we have applied to a healthy population. Two homozygotes for the N350S and 1524 + 95A-->G mutations were detected, which gives a population frequency of 2.6%. The overall frequencies of the ASA-PD mutations were shown to be 17.5% for the N350S change and 13.0% for the 1524 + 95A-->G change, estimating each mutation separately. In addition, the frequency of both PD associated mutations occurring together on the same chromosome was found to be 12.3% in our population. The study has also allowed us to establish a new control ASA activity range, which was based on assay of blood from persons who had been shown at the DNA level not to carry ASA PD associated mutations.

A single origin for the most frequent mutation causing late infantile metachromatic leucodystrophy.

Metachromatic leucodystrophy is an autosomal recessive degenerative disease of the nervous system caused by the deficiency of the lysosomal enzyme arylsulphatase A (ARSA). We report here on the high incidence of late infantile MLD among Muslim Arabs originating from Jerusalem, most probably because of a founder effect. All the patients were found to be homozygous for 459 + 1 G-->A, a mutation which destroys the splice donor site of exon 2 of the ARSA gene. This mutation has been reported to be the most common mutation causing MLD. We studied the ARSA haplotype defined by three intragenic polymorphic sites in DNA samples from Muslim Arab patients from Jerusalem, a Christian Arab patient originating from the region, and eight other white patients, all homozygous for the 459 + 1 G-->A mutation. All the alleles carried the same haplotype which is in complete linkage disequilibrium with the mutation. This finding indicates a common origin for the 459 + 1 G-->A mutation which may have been introduced into Jerusalem at the time of the Crusades.

Missense mutations in the arylsulphatase A genes of metachromatic leukodystrophy patients.

Novel predicted disease-causing mutations have been defined in three patients with metachromatic leukodystrophy (MLD). The first new mutation is a C-->A change at base 884 in exon 5 of the arylsulphatase A (ASA) gene causing a serine to tyrosine substitution at position 295 of the protein (S295Y). A late-infantile MLD patient was found to be homozygous for this mutation. The second mutation is a G-->A substitution at nucleotide 1144 in exon 7, that causes a glutamic acid to lysine substitution at amino acid 382 (E382K). A juvenile MLD patients was found to be homozygous for this mutation. Finally an adult MLD patient has been shown to be heterozygous for two novel point mutations in exon 3. These are both C-->T changes at position 635 and 671 that result in alanine to valine substitutions at amino acids 212 (A212V) and 224 (A224V) of the ASA protein.

Gaucher's disease in the United Kingdom: screening non-Jewish patients for the two common mutations.

Twenty-six patients with Gaucher's disease diagnosed in the United Kingdom and two obligate carriers, all of non-Jewish origin, were screened for the two common disease causing mutations and two rarer mutations in the glucocerebrosidase gene. These mutations are referred to as N370S, L444P, Ins84G, and 1066 + 1G-->A, respectively. The results showed that out of 54 alleles screened, 26% were N370S, 35% were L444P, and the remaining 39% were rare or undefined. The results also showed a clear correlation between the presence of at least one N370S allele and mild disease.

Prevalence of common mutations in the arylsulphatase A gene in metachromatic leukodystrophy patients diagnosed in Britain.

The frequency of two common disease-associated mutations in the arylsulphatase A (ASA) gene, and of a mutation causing ASA pseudodeficiency, have been established in metachromatic leukodystrophy patients diagnosed in our laboratory. A total of 37 mutant genes have been analysed. The G-->A change destroying the splice donor site of exon 2 is generally associated with more severe disease and was found in 43.2% of mutant ASA genes. The C-->T transition causing a proline to leucine substitution at position 426 in exon 8 (P426-->L) is associated with later onset disease, and was found in 16.2% of mutant genes. The A-->G transition leading to loss of a polyadenylation signal associated with ASA pseudodeficiency was present at a frequency of 7.5% in the patients and heterozygotes studied.

Application of a flowchart for the detection of lysosomal storage diseases in 105 high-risk Brazilian patients.

Lysosomal storage diseases (LSD) are a group of more than 40 disorders, many of them with overlapping phenotype, in which clinical diagnosis is often difficult. Definitive diagnosis is based on enzyme assays, a large number of such assays usually being necessary during the investigation of each patient. In addition, there will frequently be a need for tissue culture in order to provide enough material for analysis. Taking into account these difficulties, we designed a flowchart for the detection of LSD that is based on 2 sets of tests requiring only random urine and heparinized blood. Here we describe this routine and report the results of its application to 105 Brazilian patients in whom a LSD was suspected. We think that the application of this rationale represents a saving of work and costs, and should be of special interest to genetic centers in developing countries.

Very low levels of high density lipoprotein cholesterol in four sibs of a family with non-neuropathic Niemann-Pick disease and sea-blue histiocytosis.

Very low serum levels of high density lipoprotein cholesterol ranging from 8.6 to 13.9 mg/dl were detected in four out of 12 sibs of a Brazilian kindred with the non-neuropathic form of Niemann-Pick disease. Hepatosplenomegaly, interstitial infiltration of the lungs, absence of neurological signs, sea-blue histiocytes in the bone marrow and liver, and high values for serum acid phosphatase (18 to 32 U/l) were common to all affected children. Leucocyte acid sphingomyelinase activity ranged from 3.6 to 6.5% of mean control values, and fibroblast activity from 9 to 13% of mean controls. The parents had low-normal levels. The relationship between these findings is unclear and deserves further investigation.

Inborn errors of metabolism. Sensitivity of screening tests in high risk patients.

We estimated the sensitivity of a screening procedure (SP) for inborn errors of metabolism (IEM) in 566 referred, high-risk patients. The 143 (25.3% of the total sample) patients with initial abnormal results in at least one screening test (ST) were recalled for further investigations. An IEM was diagnosed in 40.6 percent of the 106 patients who came for reevaluation. In 114 of the remaining 423 patients who had normal initial ST, an IEM was still suspected on basis of clinical, radiological, and/or laboratory findings and was confirmed in 30 of such patients (5.3% of the total sample and 7.1% of the patients with normal results in the SP). The sensitivity of the SP was estimated maximally as 67.4 percent and the efficiency as 80.4 percent. Twenty-five of the 30 cases undetected with the SP were patients with sphingolipidoses. The simple inclusion of thin-layer chromatography of urinary oligosaccharides in the SP should allow the detection of at least one half of these cases, increasing its sensitivity by 14.1 percent and its efficiency by 4.6 percent. In at least 7.1 percent of patients with an initial normal ST, an IEM was detected. These would have remained undiagnosed if the limitations of the SP employed had not been fully understood.

Influence of aliphatic alcohols on the hepatic response to halogenated olefins.

The role of alcohols in potentiating the toxicity of halogenated hydrocarbon solvents has been reviewed. The toxicity of carbon tetrachloride and chloroform can be markedly potentiated by prior treatment with ethanol or phenobarbital. Trichloroethylene toxicity may also be potentiated by ethanol ingestion. Prior ethanol ingestion acts by altering biochemical parameters that result in an increased response to subsequent solvent exposure. Simultaneous exposure to both ethanol and trichloroethylene allows for competitive substrate inhibition of metabolism since these compounds share several common enzymatic pathways. Thus the toxic response to multiple exposures varies depending upon the time sequence and the comparative levels of the individual components. Phenobarbital apparently potentiates solvent toxocity by induction of the microsomal mixed function oxidase system. Ethanol, either on a chronic or single dose basis, also has the ability to stimulate this enzyme system. Although alteration of the microsomal mixed function oxidase system by chronic ethanol ingestion may play an important role in potentiation of solvent toxicity, the potentiation seen following a single dose of ethanol cannot be fully accounted for by the known effects of ethanol on the mixed function oxidase system. In addition to ethanol a large number of other alcohols will markedly potentiate the hepatotoxic response to solvents such as carbon tetrachloride and chloroform. The mechanisms involved in such potentiation are not known at the present time.

Experimental toxicology of pyrolysis and combustion hazards.

Data are presented on the acute toxicity (mortality only) of the thermal degradation products of polymers obtained by two methods of degradation. One system utilized a slowly increasing temperature (5 degrees C/min) and gradual degradation of the polymer with the rats being exposed to degradation products as they were evolved. In this system the more toxic polymers included wool, polypropylene, poly(vinyl chloride), and urethane foam. The second system utilized conditions of rapid combustion and exposure of rats to the total products of combustion for a period of 4 hr. In this system the more toxic materials included red oak, cotton, acrylonitrile-butadiene-styrene (ABS), and styrene-acrylonitrile. It is of interest to note that the natural product wool is among the least toxic under these rapid combustion conditions and among the most toxic under slow pyrolysis conditions. Other materials also vary in the comparative toxicity of their thermal degradation products, depending upon the conditions of degradation and animal exposure. The two experimental techniques presented here may well represent the two extreme conditions of rapid combustion versus slow pyrolysis. Intermediate types of fire situations might be expected to result in relative acute toxicities somewhere between these two extremes. This report deals with acute toxicity on the basis of mortality data only and does not include other parameters of toxicity such as organ weights and histopathology.