Anthony P. DeCaprio

Application of the Hard and Soft, Acids and Bases (HSAB) Theory to Toxicant–Target Interactions

Many chemical toxicants and/or their active metabolites are electrophiles that cause cell injury by forming covalent bonds with nucleophilic targets on biological macromolecules. Covalent reactions between nucleophilic and electrophilic reagents are, however, discriminatory since there is a significant degree of selectivity associated with these interactions. Over the course of the past few decades, the theory of Hard and Soft, Acids and Bases (HSAB) has proven to be a useful tool in predicting the outcome of such reactions. This concept utilizes the inherent electronic characteristic of polarizability to define, for example, reacting electrophiles and nucleophiles as either hard or soft. These HSAB definitions have been successfully applied to chemical-induced toxicity in biological systems. Thus, according to this principle, a toxic electrophile reacts preferentially with biological targets of similar hardness or softness. The soft/hard classification of a xenobiotic electrophile has obvious utility in discerning plausible biological targets and molecular mechanisms of toxicity. The purpose of this perspective is to discuss the HSAB theory of electrophiles and nucleophiles within a toxicological framework. In principle, covalent bond formation can be described by using the properties of their outermost or frontier orbitals. Because these orbital energies for most chemicals can be calculated using quantum mechanical models, it is possible to quantify the relative softness (σ) or hardness (η) of electrophiles or nucleophiles and to subsequently convert this information into useful indices of reactivity. This atomic level information can provide insight into the design of corroborative laboratory research and thereby help investigators discern corresponding molecular sites and mechanisms of toxicant action. The use of HSAB parameters has also been instrumental in the development and identification of potential nucleophilic cytoprotectants that can scavenge toxic electrophiles. Clearly, the difficult task of delineating molecular sites and mechanisms of toxicant action can be facilitated by the application of this quantitative approach.

Relationship of Thyroid Hormone Levels to Levels of Polychlorinated Biphenyls, Lead, p,p′- DDE, and Other Toxicants in Akwesasne Mohawk Youth

Background It is well documented that acute exposure to high levels of persistent organic pollutants, such as polychlorinated biphenyls (PCBs), p,p′-dichlorophenyldichloroethylene (p,p′-DDE), and hexachlorobenzene (HCB), can affect human health including thyroid function. Chronic exposure to multiple toxicants is common but difficult to analyze, and most prior studies have focused on adults or newborns, creating a gap in our understanding of multitoxicant effects among adolescents. Objective We investigated whether levels of PCBs, p,p′-DDE, HCB, mirex, lead, and mercury reflecting past chronic exposure are associated with alterations in levels of thyroid-stimulating hormone (TSH), triiodothyronine (T3), total thyroxine (TT4), and free thyroxine (FT4) among older children and adolescents. Methods The sample consists of youth from the Akwesasne Mohawk Nation (n = 232) who reside in proximity to several industries that have contaminated the local environment. We used multiple regression analysis to examine the effect of PCB groupings, p,p′-DDE, HCB, lead, and mercury on thyroid hormones after adjusting for sociodemographic covariates and controlling for all other toxicants. Results Exposure to PCBs affects the thyroid hormone profile in adolescents. The group of persistent PCBs was positively associated with TSH but inversely related to FT4. Nonpersistent PCBs were significantly and negatively related to FT4 only. HCB was negatively associated with T4, and lead was positively associated with T3. Breast-fed adolescents had higher levels of persistent PCBs and p,p′-DDE but not of nonpersistent PCBs or any other toxicant when compared with non-breast-fed adolescents. Though having lower levels of persistent PCBs and p,p′-DDE, non-breast-fed adolescents exhibited significant relationships between persistent PCBs and TSH and FT4, but breast-fed adolescents did not. It appears that PCBs from breast milk obscure the relationship between prenatal PCB exposure and thyroid function by adding random variation in PCB levels. Conclusion Our results demonstrate a reduction in thyroid function in adolescents in relation to their current serum levels of PCBs. These observations are consistent with the hypothesis that pre-natal exposure to PCBs alters thyroid function in a long-lasting manner but does not exclude the possibility that postnatal exposure is influential also.

Reductive and oxidative degradation of iopamidol, iodinated X-ray contrast media, by Fe(III)-oxalate under UV and visible light treatment

Iopamidol, widely employed as iodinated X-ray contrast media (ICM), is readily degraded in a Fe(III)-oxalate photochemical system under UV (350 nm) and visible light (450 nm) irradiation. The degradation is nicely modeled by pseudo first order kinetics. The rates of hydroxyl radical (OH) production for Fe(III)-oxalate/H2O2/UV (350 nm) and Fe(III)-oxalate/H2O2/visible (450 nm) systems were 1.19 ± 0.12 and 0.30 ± 0.01 μM/min, respectively. The steady-state concentration of hydroxyl radical (OH) for the Fe(III)-oxalate/H2O2/UV (350 nm) conditions was 10.88 ± 1.13 × 10(-14) M and 2.7 ± 0.1 × 10(-14) M for the Fe(III)-oxalate/H2O2/visible (450 nm). The rate of superoxide anion radical (O2(-)) production under Fe(III)-oxalate/H2O2/UV (350 nm) was 0.19 ± 0.02 μM/min with a steady-state concentration of 5.43 ± 0.473 × 10(-10) M. Detailed product studies using liquid chromatography coupled to Q-TOF/MS demonstrate both reduction (multiple dehalogenations) and oxidation (aromatic ring and side chains) contribute to the degradation pathways. The reduction processes appear to be initiated by the carbon dioxide anion radical (CO2(-)) while oxidation processes are consistent with OH initiated reaction pathways. Unlike most advanced oxidation processes the Fe(III)-oxalate/H2O2/photochemical system can initiate to both reductive and oxidative degradation processes. The observed reductive dehalogenation is an attractive remediation strategy for halogenated organic compounds as the process can dramatically reduce the formation of the problematic disinfection by-products often associated with oxidative treatment processes.

Analysis of PCB congeners related to cognitive functioning in adolescents.

To investigate the characteristics of PCBs that are linked to cognitive functioning, those congeners that were concurrently found in 271 Mohawk adolescents were grouped according to structure (dioxin-like or non-dioxin-like) and persistence (persistent or low-persistent). After the effects of the congener groups were orthogonalized, regression analyses (controlling for a number of variables found to be related to the cognitive outcomes) examined the relationship of each congener group to scores on three cognitive tests (the non-verbal Ravens Progressive Matrices, the Test of Memory and Learning, and the Woodcock Johnson-Revised). Five subtests from these cognitive tests were found to be associated with one or more PCB congener groups, most often at a moderate level. Two measures of long-term memory (Delayed Recall and Long Term Retrieval) were associated with all four congener groups. Nevertheless, examination of the role of individual congeners in the significantly related congener groups revealed that almost all congeners associated with cognitive outcomes were non-dioxin-like and ortho-substituted. A notable exception was the Ravens test where scores were associated only with dioxin-like congeners. This finding adds to the limited evidence of neurotoxic effects of dioxin-like congeners. Auditory Processing was related only to the persistent congener group. The association of the non-persistent congener group with three cognitive test scores (Delayed Recall, Long Term Retrieval and Comprehension-Knowledge) suggests that the Mohawk adolescents have experienced continuing or recent environmental exposure to PCBs that is sufficient to result in detectable cognitive decrements. Comparison of our findings with those of other human studies was limited by the relative lack of specificity of both PCB measures and cognitive outcome measures in much previous work.

Polychlorinated biphenyls in serum of the Siberian Yupik people from St. Lawrence Island, Alaska

Abstract Objectives. To determine serum levels of polychlorinated biphenyls (PCBs) in Siberian Yupik adults from St. Lawrence Island, Alaska, and to determine the relative contribution of atmospheric transport of PCBs and local contamination to body burdens. Study Design. Siberian Yupiks of various ages were recruited from three populations: residents of the village of Gambell, residents of the village of Savoonga who did not have family hunting camps near the Northeast Cape (NEC), a Formerly Used Defense Site (FUDS) known to be contaminated with PCBs, and residents of Savoonga whose families had a hunting camp at the NEC. Methods. Levels of PCBs were measured in serum samples from 130 people, ages 19–76. These Alaska Natives follow a traditional diet high in marine mammals and fish, which bioconcentrate organochlorine compounds that migrate to the Arctic via global air transport and ocean currents. Results. The lipid-adjusted serum PCB levels of those members of families with hunting camps at the NEC had a mean lipid-adjusted PCB concentration of 1,143 ppb, whereas other residents of Savoonga had values of 847 ppb and residents of Gambell had values of 785 ppb. Conclusions. Our observations suggest that atmospheric transport of PCBs contributes to levels in the Yupik people, but that the abandoned military site at the NEC may also contribute to the human body burden in those individuals who have either spent substantial time or consumed food from there. (Int J Circumpolar Health 2005; 64(4):322–335)

Relationships of Polychlorinated Biphenyls and Dichlorodiphenyldichloroethylene (p,p’-DDE) with Testosterone Levels in Adolescent Males

Background: Concern persists over endocrine-disrupting effects of persistent organic pollutants (POPs) on human growth and sexual maturation. Potential effects of toxicant exposures on testosterone levels during puberty are not well characterized. Objectives: In this study we evaluated the relationship between toxicants [polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylene (p,p´-DDE), hexachlorobenzene (HCB), and lead] and testosterone levels among 127 Akwesasne Mohawk males 10 to < 17 years of age with documented toxicant exposures. Methods: Data were collected between February 1996 and January 2000. Fasting blood specimens were collected before breakfast by trained Akwesasne Mohawk staff. Multivariable regression models were used to estimates associations between toxicants and serum testosterone, adjusted for other toxicants, Tanner stage, and potential confounders. Results: The sum of 16 PCB congeners (Σ16PCBs) that were detected in ≥ 50% of the population was significantly and negatively associated with serum testosterone levels, such that a 10% change in exposure was associated with a 5.6% decrease in testosterone (95% CI: –10.8, –0.5%). Of the 16 congeners, the more persistent ones (Σ8PerPCBs) were related to testosterone, whereas the less persistent ones, possibly reflecting more recent exposure, were not. When PCB congeners were subgrouped, the association was significant for the sum of eight more persistent PCBs (5.7% decrease; 95% CI: –11, –0.4%), and stronger than the sum of six less persistent congeners (3.1% decrease; 95% CI: –7.2, 0.9%). p,p´-DDE was positively but not significantly associated with serum testosterone (5.2% increase with a 10% increase in exposure; 95% CI: –0.5, 10.9%). Neither lead nor HCB was significantly associated with testosterone levels. Conclusions: Exposure to PCBs, particularly the more highly persistent congeners, may negatively influence testosterone levels among adolescent males. The positive relationship between p,p´-DDE and testosterone indicates that not all POPs act similarly. Citation: Schell LM, Gallo MV, Deane GD, Nelder KR, DeCaprio AP, Jacobs A; Akwesasne Task Force on the Environment. 2014. Relationships of polychlorinated biphenyls and dichlorodiphenyldichloroethylene (p,p´-DDE) with testosterone levels in adolescent males. Environ Health Perspect 122:304–309; http://dx.doi.org/10.1289/ehp.1205984

γ-Diketone Axonopathy: Analyses of Cytoskeletal Motors and Highways in CNS Myelinated Axons

2,5-Hexanedione (HD) intoxication is associated with axon atrophy that might be responsible for the characteristic gait abnormalities, hindlimb skeletal muscle weakness and other neurological deficits that accompany neurotoxicity. Although previous mechanistic research focused on neurofilament triplet proteins (NFL, NFM, NFH), other cytoskeletal targets are possible. Therefore, to identify potential non-NF protein targets, we characterized the effects of HD on protein-protein interactions in cosedimentation assays using microtubules and NFs prepared from spinal cord of rats intoxicated at different daily dose rates (175 and 400 mg/kg/day). Results indicate that HD did not alter the presence of alpha- or beta-tubulins in these preparations, nor were changes noted in the distribution of either anterograde (KIF1A, KIF3, KIF5) or retrograde (dynein) molecular motors. The cosedimentation of dynactin, a dynein-associated protein, also was not affected. Immunoblot analysis of microtubule-associated proteins (MAPs) in microtubule preparations revealed substantial reductions (45-80%) in MAP1A, MAP1B heavy chain, MAP2, and tau regardless of HD dose rate. MAP1B light chain content was not altered. Finally, HD intoxication did not influence native NF protein content in either preparation. As per previous research, microtubule and NF preparations were enriched in high-molecular weight NF species. However, these NF derivatives were common to both HD and control samples, suggesting a lack of pathognomonic relevance. These data indicate that, although motor proteins were not affected, HD selectively impaired MAP-microtubule binding, presumably through adduction of lysine residues that mediate such interactions. Given their critical role in cytoskeletal physiology, MAPs could represent a relevant target for the induction of gamma-diketone axonopathy.

Diabetes Prevalence in Relation to Serum Concentrations of Polychlorinated Biphenyl (PCB) Congener Groups and Three Chlorinated Pesticides in a Native American Population.

Background: Exposure to persistent organic pollutants (POPs) is known to increase risk of diabetes. Objective: To determine which POPs are most associated with prevalence of diabetes in 601 Akwesasne Native Americans. Methods: Multiple logistic regression analysis was used to assess associations between quartiles of concentrations of 101 polychlorinated biphenyl (PCBs) congeners, congener groups and three chlorinated pesticides [dichlorodiphenyldichloroethylene (DDE), hexachlorobenzene (HCB) and mirex] with diabetes. In Model 1, the relationship between quartiles of exposure and diabetes were adjusted only for sex, age, body mass index (BMI), and total serum lipids. Model 2 included additional adjustment for either total PCBs or total pesticides. Results: Total serum PCB and pesticide concentrations were each significantly associated with prevalence of diabetes when adjusted only for covariates (Model 1), but neither showed a significant OR for highest to lowest quartiles after additional adjustment for the other (Model 2). When applying Model 2 to PCB congener groups and individual pesticides, there were significant omnibus differences between the four quartiles (all ps < 0.042) for most groups, with the exception of penta- and hexachlorobiphenyls, DDE and mirex. However, when comparing highest to lowest quartiles only non- and mono-ortho PCBs [OR = 4.55 (95% CI: 1.48, 13.95)], tri- and tetrachloro PCBs [OR = 3.66 (95% CI: 1.37, –9.78)] and HCB [OR = 2.64 (95% CI: 1.05, 6.61)] showed significant associations with diabetes. Among the non- and mono-ortho congeners, highest to lowest quartile of dioxin TEQs was not significant [OR = 1.82 (95% CI: 0.61, 5.40)] but the OR for the non-dioxin-like congeners was [OR = 5.01 (95% CI: 1.76, 14.24)]. Conclusion: The associations with diabetes after adjustment for other POPs were strongest with the more volatile, non-dioxin-like, low-chlorinated PCB congeners and HCB. Because low-chlorinated congeners are more volatile, these observations suggest that inhalation of vapor-phase PCBs is an important route of exposure. Citation: Aminov Z, Haase R, Rej R, Schymura MJ, Santiago-Rivera A, Morse G, DeCaprio A, Carpenter DO, and the Akwesasne Task Force on the Environment. 2016. Diabetes prevalence in relation to serum concentrations of polychlorinated biphenyl (PCB) congener groups and three chlorinated pesticides in a Native American population. Environ Health Perspect 124:1376–1383; http://dx.doi.org/10.1289/ehp.1509902

Covalent adduction of nitrogen mustards to model protein nucleophiles.

Protein adducts have the potential to serve as unique biomarkers of exposure to compounds of interest. Many xenobiotics (or their metabolites) are electrophilic and therefore reactive with nucleophilic amino acid residues on proteins. Nitrogen mustards are reactive xenobiotics with potential use as chemical warfare agents (CWA) or agents of terrorist attack, in addition to being employed as chemotherapeutic agents. The present study utilized cysteine-, lysine-, and histidine-containing model peptides to characterize in vitro adduction of the nitrogen mustards mechloroethamine (HN-2) and tris-(2-chlorethyl)amine (HN-3) to these nucleophilic amino acid residues by means of liquid chromatography-tandem mass spectrometry. The study assessed the structure of adducts formed, the time course of adduct formation, concentration-response relationships, and temporal stability of adducts. Adduction was hypothesized to occur on all three model peptides via initial formation of a reactive aziridinium intermediate for both mechloroethamine and tris-(2-chlorethyl)amine, followed by covalent adduction to nucleophilic residues. While adduction was found to occur most readily with cysteine, it was also observed at lysine and histidine, demonstrating that adduction by mechloroethamine and tris-(2-chlorethyl)amine is possible at multiple nucleophilic sites. Following solid phase extraction cleanup, adducts formed with mechloroethamine were stable for up to three weeks. Adducts formed with tris-(2-chlorethyl)amine were less stable; however, hydrolyzed secondary adducts were observed throughout the three week period. This study demonstrates that the nitrogen mustards mechloroethamine and tris-(2-chlorethyl)amine form stable adducts with reactive protein nucleophiles other than cysteine.

Comparative covalent protein binding of 2,5-hexanedione and 3-acetyl-2,5-hexanedione in the rat.

2,5-Hexanedione (HD) is the metabolite implicated in n-hexane neurotoxicity. This γ-diketone reacts with protein lysine amines to form 2,5-dimethylpyrrole adducts. Pyrrole adduction of neurofilaments (NF) and/or other axonal proteins was proposed as a critical step in the neuropathy. While pyrrole adduction is widely accepted as necessary, subsequent pyrrole oxidation, which may result in protein cross-linking, was alternatively postulated as the critical mechanistic step. Previous studies have indicated that 3-acetyl-2,5-HD (AcHD), an analogue that forms pyrroles that do not oxidize, was not neurotoxic in rats. However, relative levels of pyrrole adduction of NF or other axonal proteins were not reported. In the present study, groups of 6 male Wistar rats were given saline, [1,6-14C]-HD (3 mmol/kg/d), or [5-14C]-AcHD (0.1 mmol/kg/d), i.p. for 21 d. HD- and AcHD-treated rats lost 10% and gained 14% body weight, respectively, compared to a 22% gain for control rats. At termination, HD- and AcHD-treated rats exhibited mean scores of 3.5 and 1.4, respectively, for hindlimb weakness (0–5 scale). Incorporation of radiolabel from HD was 27.8 ± 3.9, 13.9 ± 2.6, and 7.8 ± 0.6 nmol/mg in plasma protein, purified globin, and axonal cytoskeletal proteins, respectively, compared to 0.6 ± 0.1, 1.6 ± 0.5, and 1.0 ± 0.1 for AcHD. Binding of HD to the NF-L, -M, and -H subunit proteins from treated animals was 4-, 24-, and 13-fold higher, respectively, that that of AcHD, indicating differing stoichiometry and patterns of NF adduction for the two diketones. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of globin and NF proteins did not demonstrate protein cross-linking for either diketone at the dose levels and time period examined. These results indicate that that the lack of neurotoxicity previously reported for AcHD may reflect differences in adduct levels at critical axonal target sites rather than an inability to form cross-linking adducts. Based on these data, further studies are required to fully assess the neurotoxic potency of AcHD and other non-cross-linking analogues as compared to HD.