Clark D. Carrington

Update of US FDA's Total Diet Study food list and diets.

The US Food and Drug Administrations (FDA) Total Diet Study (TDS) has been conducted continuously since the early 1960s to measures levels of various pesticide residues, contaminants, and nutrients in foods and to estimate the dietary exposures to these compounds. Both the TDS food list and the consumption amounts used for estimating exposures are based on results of nationwide food consumption surveys, and they are updated periodically to reflect changes in food consumption patterns. The most recent update was completed in 2003 using the same methodology employed in the previous update (1990). The updated food list includes approximately the same number of foods (285) as the previous list (290). Although most (75%) foods are the same in both versions, the new list reflects trends in consumption of foods containing less fat. The updated diets reflect an increase in total food consumption, with most notable increases in consumption of grains and beverages. A case study comparing cadmium exposures calculated from both the 1990 and 2003 versions of the TDS demonstrated the potential impact of changes in both the food list and consumption amounts on TDS exposure estimates.

In vitro binding of [14C]acrylamide to neurofilament and microtubule proteins of rats

Acrylamide produces a dying back type of neuropathy in which there is an accumulation of neurofilaments in the axons. The in vitro binding of [14C]acrylamide to neurofilament and microtubule proteins obtained from rat spinal cord and brain was investigated. The relative binding to the high and middle molecular weight neurofilament was greater than to the low molecular weight neurofilament, while the rate of binding to MAP-1 (microtubule associated protein-1) and -2 was much greater than to tubulin. The binding rate to a 53 kDa protein which co-purified with the neurofilaments was between those of the middle and high molecular weight neurofilaments while the lowest rate of binding was to glial fibrillary acidic protein. These data indicate that there is a direct binding of acrylamide to cytoskeletal proteins.

Axoplasmic Transport and Turnaround of Neurotoxic Esterase in Hen Sciatic Nerve

Abstract: We have recently found that there is a proximo‐distal delay in the recovery of neurotoxic esterase (NTE) following inhibition along the sciatic nerve of the hen. To determine whether this delay could be due to a requirement for the transport of newly synthesized NTE from the cell body, we investigated the transport of NTE by measuring the rate of accumulation of activity at either one or two ligations. Although rapid turnaround of accumulated protein confounds calculation of the transport rate, it appeared that NTE is transported down the hen sciatic nerve at a rate close to 300 mm/day. Acetylcholinesterase (AChE) was found to be transported at a rate of about 500 mm/day, which is close to the expected rate of fast axoplasmic transport in the chicken. The relatively rapid turnaround of NTE compared with the retrograde transport rate precluded the estimation of a retrograde transport rate. A model is presented that accounts for turnaround as a result of exchange between mobile and stationary transport pools. Exchange of NTE between pools may account for the rapid turnaround of NTE described in this paper and for the proximo‐distal delay in recovery as a dilution of newly synthesized NTE in the anterograde fast transport pool by inhibited protein as it travels down the nerve.

Prophylaxis and the mechanism for the initiation of organophosphorous compound-induced delayed neurotoxicity

Recent work concerned with the mechanism underlying the development of organophosphorous compound-induced delayed neurotoxicity (OPIDN) is reviewed. Topics covered include the prophylaxis of OPIDN by phenylmethylsulfonyl fluoride and other agents, neurotoxic esterase (NTE) as measured using either labelled diisopropyl phosphorofluoridate or an esterase assay, and the relationship between NTE and the development of OPIDN. There is considerable evidence that NTE has the biochemical properties which should be expected for the initiation site for OPIDN. However, the in vitro assays as currently performed may not entirely reflect the behavior of organophosphorous compounds in vivo, or the assays may not be sensitive enough to identify the actual target. It is argued that prophylaxis is a distinguishing characteristic of OPIDN which is not necessarily related to NTE inhibition, although it does provide evidence that NTE is involved. It is concluded that the NTE hypothesis could be furthered by additional studies with peripheral nerve, more sensitive methods for the detection of potential binding sites, and the establishment of a physiological role for NTE which relates it to the neuropathy.

The correlation between the recovery rate of neurotoxic esterase activity and sensitivity to organophosphorus-induced delayed neurotoxicity.

Neurotoxic esterase (NTE) has been proposed to be the initiation site of organophosphorus compound-induced delayed neurotoxicity (OPIDN). There are two apparent problems associated with this hypothesis: NTE activity in the brain returns to nearly normal levels before the onset of the neuropathy, and NTE is present in and inhibited by organophosphorus compounds in young animals and other species which are relatively insensitive to the neurotoxic effects of these compounds. This paper presents data suggesting that differences in the recovery rates of NTE activity may account for some of these discrepancies. First, the onset of recovery of NTE activity following sc administration of 1.7 mg/kg of O,O-diisopropylphosphorofluoridate (DFP) in the hen sciatic nerve occurred several days later than in the brain. Furthermore, recovery was slower in distal than proximal parts of the nerve. This information indicates that NTE activity is depressed for a longer period at the site of the neuropathy than it would appear from the measurement of NTE activity in brain. Second, the rate of recovery of NTE activity was faster in the brains of chicks, of rats, and of hens treated with a daily po dose of 15 mg/kg cortisone acetate than it was in untreated hens. However, there was no significant increase in the NTE recovery rate in the peripheral nerves of the chicks or the cortisone-treated hens. Thus, it appears that although slower distal recovery could account for the greater sensitivity of longer axons to OPIDN, other factors are operating in chicks and cortisone-treated hens.

Paraoxon reversibly inhibits neurotoxic esterase

It has recently been reported that two paraoxon-insensitive carboxylesterases may be distinguished by their sensitivity to mipafox. However, we have not been able to reliably detect two components under the conditions of the widely used assay for neurotoxic esterase (NTE). We have determined that this discrepancy is a result of differences in the technique of preinhibition by paraoxon and mipafox. We report here that paraoxon is apparently able to reduce the rate of inhibition of both neurotoxic esterase isozymes by mipafox in a concentration-dependent manner. As a result, the rate of inhibition of NTE by mipafox is greater when sequential, rather than concurrent, preinhibition is utilized. An apparently greater reduction in the inhibition rate of the more sensitive minor component may make the two isozyme species indistinguishable in the presence of paraoxon at concentrations at or above 40 microM.

Induction of hepatic microsomal cytochrome P-450 and inhibition of brain, liver, and plasma esterases by an acute dose of S,S,S-tri-n-butyl phosphorotrithioate (DEF) in the adult hen

The differential effects of oral and dermal administration of single doses of 100 to 1000 mg/kg S,S,S-tri-n-butyl phosphorotrithioate (DEF) on nonspecific esterases and liver metabolism enzymes were investigated one day following administration. O,O-Diethyl O-(4-nitrophenyl) phosphorothioate (parathion) and tri-o-cresyl phosphate (TOCP) were used as negative and positive controls for organophosphorus-induced delayed neurotoxicity (OPIDN). Brain acetylcholinesterase was significantly inhibited with topical doses of 500 and 1,000 mg/kg of DEF and with orally and dermally applied parathion. Plasma cholinesterase and liver microsomal carboxylesterase activities were significantly reduced from control in all treatment groups. Neurotoxic esterase (NTE) was significantly decreased from control with topical dosing of 200, 500, and 1000 mg/kg DEF and with TOCP treatments. Oral doses of DEF increased cytochrome P-450 content by 70 to 200% while dermal application caused a 200 to 325% increase over control. p-Chloro-N-methylaniline demethylase was also increased by DEF treatments but to a lesser extent than that of aniline hydroxylase or cytochrome P-450 content. TOCP and parathion had no significant effect on liver microsomal oxidative enzymes. Liver microsomal proteins from hens treated with phenobarbital (PB), 3-methylcholanthrene (3MC), or DEF were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A striking increase in a 49K protein band in microsomes from PB and DEF (616 and 338%, respectively) treated hens was seen, while the 55K protein band showed an 861% increase in microsomes from 3MC-treated hens. In conclusion, dermally applied DEF was more effective in inhibiting esterases and inducing cytochrome P-450 than orally administered DEF; toxicity was directly related to the dose and route of administration.

The time course of protection from delayed neurotoxicity induced by tri-o-cresyl phosphate and o,o-diisopropyl phosphorofluoridate by phenyl methyl sulfonyl fluoride in chickens

The time dependence of the ability of phenyl methyl sulfonyl fluoride (PMSF) to protect adult hens from developing signs of paralysis following the administration of 750 mg/kg tri-ortho-cresyl phosphate (TOCP), p.o., of 1.7 mg/kg O,O-diisopropyl phosphorofluoridate (DFP), s.c., was investigated. PMSF was able to protect the hens from organophosphorus-induced delayed neurotoxicity (OPIDN) when given between 1 and 24 h before the administration of TOCP, or when given 4 h before DFP. However, PMSF was ineffective at preventing paralysis when given 24 h following dosing with TOCP or when given later than 4 h before DFP administration. These results support the notion that PMSF acts at the same site as the organophosphorus esters.

Triphenyl phosphite neurotoxicity in the hen: inhibition of neurotoxic esterase and of prophylaxis by phenylmethylsulfonyl fluoride

The neuropathic syndrome resulting in the cat and the rat from single or multiple doses of the phosphorous acid ester tiphenyl phosphite (TPP) has been reported to differ from the syndrome caused by numerous phosphoric acid esters, which is known as organophosphorous compound-induced delayed neurotoxicity (OPIDN). Since the hen is used to test compounds for OPIDN, we chose to study the neurotoxicity of single subcutaneous doses of TPP using this animal model. TPP (1000 mg/kg) produced progressive ataxia and paralysis which began to develop 5–10 days after dosing. Similar signs were observed when subcutaneous doses of the OPIDN-causing agents tri-o-cresyl phosphate (TOCP) or diisopropyl phosphorofluoridate (DFP) were administered. The minimum neurotoxic dose of TPP was 500 mg/kg. Prior administration of phenylmethylsulfonyl fluoride (PMSF) prevented the development of a neuropathy induced by DFP, but did not fully protect the hens from TPP or TOCP. PMSF slowed, but did not prevent, the neuropathy caused by TOCP. PMSF reduced the neurotoxicity of 500 mg/kg TPP, but increased the neurotoxicity of 1000 mg/kg TPP. TPP was found to be a very potent inhibitor of neurotoxic esterase (NTE), the putative target site for OPIDN, in vitro, with a ki of about 2.1×105 M−1min−1. Equimolar doses of either TPP (1000 mg/kg) and TOCP (1187 mg/kg) caused over 80% inhibition of neurotoxic esterase (NTE) in brain and sciatic nerve. This high level of NTE inhibition persisted for several weeks. This prolonged inhibition probably accounts for the inability of PMSF to block the neurotoxicity of TOCP. The dose-response curve for NTE inhibition 48 h after dosing indicated that a level of 70% inhibition correlated with the neurotoxicity of TPP.Subneurotoxic doses of TPP and DFP were found to have an additive effect which could be blocked by PMSF. These results indicate that TPP can cause OPIDN in the hen. The synergism between PMSF and the higher dose of TPP suggests the presence of a second neurotoxic effect as well.

In vivo binding of [14C]acrylamide to proteins in the mouse nervous system

The labeling of mouse brain and spinal cord proteins in vivo following the administration of 200 ?g/kg [(14)C]acrylamide (25 mCi/kg, i.p.) was measured by polyacrylamide gel electrophoresis and autoradiography. Several cytoskeletal proteins had the highest specific activity. These included the medium (130 kDa) and high (180 kDa) molecular weight neurofilament proteins and unknown proteins with molecular weights of 31, 33 and 57 kDa. About 0.05% of the neurofilament proteins were labeled with the dose employed. Although accurate assessments of the stoichiometry for acrylamide binding to 31, 33 and 57 kDa bands could not be made due to the low concentrations of these proteins, it is estimated that over 0.1% of these proteins bound acrylamide. There were significant amounts of acrylamide bound to proteins seven days after exposure, with labeling ranging from 15 to 60% of the levels observed one day after exposure. These results show that acrylamide binds to several major cytoskeletal proteins present in the mouse nervous system. The formation of these adducts may play a role in the neurotoxicity of acrylamide.