Jerry N. Blancato

Physicochemical and Biological Data for the Development of Predictive Organophosphorus Pesticide QSARs and PBPK/PD Models for Human Risk Assessment

ABSTRACT A search of the scientific literature was carried out for physiochemical and biological data [i.e., IC50, LD50, Kp (cm/h) for percutaneous absorption, skin/water and tissue/blood partition coefficients, inhibition ki values, and metabolic parameters such as Vmax and Km] on 31 organophosphorus pesticides (OPs) to support the development of predictive quantitative structure–activity relationship (QSAR) and physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models for human risk assessment. Except for work on parathion, chlorpyrifos, and isofenphos, very few modeling data were found on the 31 OPs of interest. The available percutaneous absorption, partition coefficients and metabolic parameters were insufficient in number to develop predictive QSAR models. Metabolic kinetic parameters (Vmax, Km) varied according to enzyme source and the manner in which the enzymes were characterized. The metabolic activity of microsomes should be based on the kinetic activity of purified or cDNA-expressed cytochrome P450s (CYPs) and the specific content of each active CYP in tissue microsomes. Similar requirements are needed to assess the activity of tissue A- and B-esterases metabolizing OPs. A limited amount of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CaE) inhibition and recovery data were found in the literature on the 31 OPs. A program is needed to require the development of physicochemical and biological data to support risk assessment methodologies involving QSAR and PBPK/PD models.

Incorporation of biological information in cancer risk assessment: Example — Vinyl chloride

Vinyl chloride (VC) is used as an example to demonstrate how biological information can be incorporated into quantitative risk assessment. The information included is the pharmacokinetics of VC in animals and humans and the data-generated hypothesis that VC primarily affects the initiation stage of the multistage carcinogenesis. The emphasis in this paper is on the improvement of risk assessment methodology rather than the risk assessment of VC per se.Sufficient data are available to construct physiologically-based pharmacokinetic models for both animals and humans. These models are used to calculate the metabolized dose corresponding to exposure scenarios in animals and in humans.On the basis of the data on liver angiosarcomas and carcinomas in rats, the cancer risk per unit of metabolized dose is comparable, irrespective of routes (oral or inhalation) of exposure. The tumor response from an intermittent/partial lifetime exposure is shown to be consistent with that from a lifetime exposure when VC is assumed to affect the first (initiation) stage of the multistage carcinogenic process. Furthermore, the risk estimates calculated on the basis of animal data are shown to be consistent with the human experience.

Parameters for Pyrethroid Insecticide QSAR and PBPK/PD Models for Human Risk Assessment

In this review we have examined the status of parameters required by pyrethroid QSAR-PBPK/PD models for assessing health risks. In lieu of the chemical,biological, biochemical, and toxicological information developed on the pyrethroids since 1968, the finding of suitable parameters for QSAR and PBPK/PD model development was a monumental task. The most useful information obtained came from rat toxicokinetic studies (i.e., absorption, distribution, and excretion), metabolism studies with 14C-cyclopropane- and alcohol-labeled pyrethroids, the use of known chiral isomers in the metabolism studies and their relation to commercial products. In this review we identify the individual chiralisomers that have been used in published studies and the chiral HPLC columns available for separating them. Chiral HPLC columns are necessary for isomer identification and for developing kinetic values (Vm,, and Kin) for pyrethroid hydroxylation. Early investigators synthesized analytical standards for key pyrethroid metabolites, and these were used to confirm the identity of urinary etabolites, by using TLC. These analytical standards no longer exist, and muste resynthesized if further studies on the kinetics of the metabolism of pyrethroids are to be undertaken.In an attempt to circumvent the availability of analytical standards, several CYP450 studies were carried out using the substrate depletion method. This approach does not provide information on the products formed downstream, and may be of limited use in developing human environmental exposure PBPK/PD models that require extensive urinary metabolite data. Hydrolytic standards (i.e., alcohols and acids) were available to investigators who studied the carboxylesterase-catalyzed hydrolysis of several pyrethroid insecticides. The data generated in these studies are suitable for use in developing human exposure PBPK/PD models.Tissue:blood partition coefficients were developed for the parent pyrethroids and their metabolites, by using a published mechanistic model introduced by Poulin and Thiele (2002a; b) and log DpH 7.4 values. The estimated coefficients, especially those of adipose tissue, were too high and had to be corrected by using a procedure in which the proportion of parent or metabolite residues that are unbound to plasma albumin is considered, as described in the GastroPlus model (Simulations Plus, Inc.,Lancaster, CA). The literature suggested that Km values be adjusted by multiplying Km by the substrate (decimal amount) that is unbound to microsomal or CYPprotein. Mirfazaelian et al. (2006) used flow- and diffusion-limited compartments in their deltamethrin model. The addition of permeability areas (PA) having diffusion limits, such as the fat and slowly perfused compartments, enabled the investigators to bring model predictions in line with in vivo data.There appears to be large differences in the manner and rate of absorption of the pyrethroids from the gastrointestinal tract, implying that GI advanced compartmental transit models (ACAT) need to be included in PBPK models. This is especially true of the absorption of an oral dose of tefluthrin in male rats, in which 3.0-6.9%,41.3-46.3%, and 5.2-15.5% of the dose is eliminated in urine, feces, and bile,respectively (0-48 h after administration). Several percutaneous studies with the pyrethroids strongly support the belief that these insecticides are not readily absorbed, but remain on the surface of the skin until they are washed off. In one articular study (Sidon et al. 1988) the high levels of permethrin absorption through the forehead skin (24-28%) of the monkey was reported over a 7- to 14-days period.Wester et al. (1994) reported an absorption of 1.9% of pyrethrin that had been applied to the forearm of human volunteers over a 7-days period.SAR models capable of predicting the binding of the pyrethroids to plasma and hepatic proteins were developed by Yamazaki and Kanaoka (2004), Saiakhov et al. (2000), Colmenarejo et al. (2001), and Colmenarejo (2003). QikProp(Schrodinger, LLC) was used to obtain Fu values for calculating partition coefficients and for calculating permeation constants (Caco-2, MDCK, and logBBB). ADMET Predictor (Simulations Plus Inc.) provided Vm~,x and Km values for the hydroxylation of drugs/pyrethroids by human liver recombinant cytochrome P450 enzymes making the values available for possible use in PBPK/PD models.The Caco-2 permeability constants and CYP3A4 Vmax and Km values are needed in PBPK/PD models with GI ACAT sub models. Modeling work by Chang et al.(2009) produced rate constants (kcat) for the hydrolysis of pyrethroids by rat serumcarboxylesterases. The skin permeation model of Potts and Guy (1992) was used topredict K, values for the dermal absorption of the 15 pyrethroids.The electrophysiological studies by Narahashi (1971) and others (Breckenridgeet al. 2009; Shafer et al. 2005; Soderlund et al. 2002; Wolansky and Harrill 2008)demonstrated that the mode of action of pyrethroids on nerves is to interfere with the changes in sodium and potassium ion currents. The pyrethroids, being highly lipid soluble, are bound or distributed in lipid bilayers of the nerve cell membrane and exert their action on sodium channel proteins. The rising phase of the action potential is caused by sodium influx (sodium activation), while the falling phase is caused by sodium activation being turned off, and an increase in potassium efflux(potassium activation). The action of allethrin and other pyrethroids is caused by an inhibition or block of the normal currents. An equation by Tatebayashi and Narahashi (1994) that describes the action of pyrethroids on sodium channels was found in the literature. This equation, or some variation of it, may be suitable for use in the PD portion of pyrethroid PBPK models.

Parameters for Carbamate Pesticide QSAR and PBPK/PD Models for Human Risk Assessment

Our interest in providing parameters for the development of quantitative structure physiologically based pharmacokinetic/pharmacodynamic (QSPBPK/PD) models for assessing health risks to carbamates (USEPA 2005) comes from earlier work with organophosphorus (OP) insecticides (Knaak et al. 2004). Parameters specific to each carbamate are needed in the construction of PBPK/PD models along with their metabolic pathways. Parameters may be obtained by (1) development of QSAR models, (2) collecting pharmacokinetic data, and (3) determining pharmacokinetic parameters by fitting to experimental data. The biological parameters are given in Table 1 (Blancato et al. 2000). Table 1 Biological Parameters Required for Carbamate Pesticide Physiologically Based Pharmacokinetic/Pharmacodynamic (PBPK/PD) Models.(a).

Aluminum interaction with human brain tau protein phosphorylation by various kinases.

Phosphorylation is an indispensable process for energy and signal transduction in biological systems. AlCl3 at 10 nM to 10 microM range activated in-vitro [gamma-32P]ATP phosphorylation of the brain (tau) tau protein in both normal human or E. coli expressed tau forms; in the presence of the kinases P34, PKP, and PKC. However, higher concentrations of ALCl3 inhibited the tau phosphorylation with P34, PKP, and PKC to a maximum at 1 mM level. AlCl3 at 100 microM to 500 microM range induced non-enzymatic phosphorylation of tau with gamma-ATP, gamma-GTP, and alpha-GTP. AlCl3 activated histone phosphorylation by P34 in a similar pattern. The hyperphosphorylation of tau by Al3+ was accompanied by molecular shift and mobility retardation in SDS-PAGE. This may demonstrate the mechanism of the longterm neurological effect of Al3+ in human brain leading to the formation of the neurofibrillary tangles related to Alzheimers disease.

Malathion disposition in dermally and orally treated rats and its impact on the blood serum acetylcholine esterase and protein profile

14C-methoxy-malathion with either pure or 50% E.C. formulated malathion were applied orally or dermally at one tenth of their LD50 to two batches of male albino rats. More than 90% of 14C was released with urine after 24 hours. The rest of 14C was detected in the feces, blood, intestines, liver and kidney in a descending order. No significant 14C was detected in other organs. Comparing the oral pure and formulated malathion treatments, there was no significant variation in the rate of disposition or excretion of 14C-malathion. However, the dermal treatment revealed that the 14C-formulated malathion was released faster than the pure one in urine in the first 24 hours; while the 14C-pure malathion showed relatively higher levels in the feces and blood in the first 24 hours. In a third batch of male albino rats, the effect of the same level of dermal treatment by either pure or 50% E.C. formulated malathion on serum acetylcholine-esterase (A. Ch. E.) activity and serum protein profile was studied. The serum A. Ch. E. activity was found to be inhibited to 40% activity after 6 to 24 hours for both treatments. However, after 96 hours the serum of the pure malathion treated rats showed full recovery of A.Ch.E. activity, while the formulated malathion treated showed only 60% activity. The SDS-PAGE analysis showed a differentiation in the serum protein bands of the 48 hours exposed rats to formulated malathion which was confirmed by the scanned gel profile. The FPLC integrated chromatograms proved an initiation of a new protein band accompanied with rearrangement of the albumin and pre-albumin bands. Thus it can be concluded that, the impact on the blood serum protein profile and A. Ch. E. activity can be used as reliable criteria to detect acute toxicity of malathion and other choline-esterase inhibitors in exposed field workers. Further research is needed to elucidate the specificity and sensitivity of such criteria as biomarkers for human exposure.

Decision model for biomarkers of exposure

The quantitation of exposure is one of the most critical elements of the risk assessment process. Contemporary risk assessment models rely on monitoring the target chemical in various environmental media and incorporate a variety of assumptions to estimate this. Recently, biomarkers have been advanced to improve estimates of the delivered dose of a given chemical or mixture of chemicals. A biomarker of exposure may be defined as an indicator of exposure to an environmental pollutant within the exposed organism, which becomes manifest before any adverse effects are evident. Measurement of such a biomarker within a biological system may potentially be used to identify and quantitate directly exposure to hazardous substances. This paper describes a decision model for the selection, evaluation, and validation of a biomarker of exposure.

Gamma aminobutyric acid radioreceptor assay: A confirmatory quantitative assay for toxaphene in environmental and biological samples

Abstract Toxaphene is a complex mixture of polychlorinated monoterpenes. Before its ban in 1982, it was the most heavily used insecticide with a cumulative world use of 409,000 metric tons. Toxaphene was found to be acutely and chronically toxic to aquatic and wild life and poses a carcinogenic risk to humans. Although the use of toxaphene has been severely limited or eliminated, it is still found in the environment due to its relative persistence with an estimated half life time of about 10 years in soils. Residue analysis of toxaphene in environmental and biological samples rely mostly on instrumental analysis such as gas chromatography (GC) and gas chromatography with mass spectrometry. These require extensive sample clean up. These instrumental analyses still have a problem of interference from other chlorinated hydrocarbons, mainly PCBs and DDT metabolites, and may not detect environmentally altered toxaphene products. Although toxaphene is a mixture of more than 200 isomers its neurotoxicity is only attributed to few isomers with a mode of action through binding to the chloride channel of the gamma-aminobutyric acid (GABA) receptor ionophore complex. [35S] tertiary butylbicyclophosphorothionate (TBPS) with specific activity higher than 60 Ci/mmole has a high binding affinity to the same sites and is now commercially available and can be used to label the GABA receptor for the development of radioreceptor assay technique. The GABA receptor was prepared by a sequence of ultra centrifugation and dialysis of mammalian (rats, cows, catfish and goats) brain homogenates. The receptor is then labeled with [35S] TBPS and the assay was conducted by measuring the displacement of radioactivity following incubation with the sample containing the analytes. The assay is fast, sensitive and requires very little or no sample preparation prior to the analysis.

Gamma aminobutyric acid radioreceptor‐assay a possible biomarker for human exposure to certain agrochemicals

Cyclodiene insecticides, hexachlorocyclohexanes, pyrethroids, bicyclophosphates, the bicycloorthocarboxylate insecticides and some of their metabolites and environmental degradation products are central nervous system toxicants with high specific binding affinity to the chloride channel of the gamma-aminobutyric acid (GABA)A receptor-ionophore sites. [35S] tertiary-butylbicyclophosphorothionate (TBPS) with specific activity higher than 60 Ci/mmole has a high binding affinity to the same sites and is now commercially available and can be used to label the GABAA receptor for the development of a radioreceptor assay technique. The GABA receptor was prepared by ultra centrifugation and dialysis of brain homogenates of either cow, goat, rat or catfish. The receptor was then labeled with [35S] TBPS and the assay was conducted by measuring the displacement of radioactivity following incubation with samples containing the analytes. A radioreceptor assay protocol was developed to measure the amount of the alpha-endosulfan in blood samples. The assay was extremely sensitive, and can detect 0.2 nM of endosulfan at a level equivalent to 0.08 ppb or 8 x 10(-11) gm of endosulfan in each ml of the blood samples.