Differences In Relative Potency Research Articles

Monocrotaline-induced liver toxicity in rat predicted by a combined in vitro physiologically based kinetic modeling approach

The aim of the present study was to use an in vitro–in silico approach to predict the in vivo acute liver toxicity of monocrotaline and to characterize the influence of its metabolism on its relative toxic potency compared to lasiocarpine and riddelliine. In the absence of data on acute liver toxicity of monocrotaline upon oral exposure, the predicted dose–response curve for acute liver toxicity in rats and the resulting benchmark dose lower and upper confidence limits for 10% effect (BMDL10 and BMDU10) were compared to data obtained in studies with intraperitoneal or subcutaneous dosing regimens. This indicated the predicted BMDL10 value to be in line with the no-observed-adverse-effect levels (NOAELs) derived from availabe in vivo studies. The predicted BMDL10–BMDU10 of 1.1–4.9 mg/kg bw/day also matched the oral dose range of 1–3 mg PA/kg bw/day at which adverse effects in human are reported. A comparison to the oral toxicity of the related pyrrolizidine alkaloids (PAs) lasiocarpine and riddelliine revealed that, although in the rat hepatocytes monocrotaline was less toxic than lasiocarpine and riddelliine, due to its relatively inefficient clearance, its in vivo acute liver toxicity was predicted to be comparable. It is concluded that the combined in vitro-PBK modeling approach can provide insight in monocrotaline-induced acute liver toxicity in rats, thereby filling existing gaps in the database on PA toxicity. Furthermore, the results reveal that the kinetic and metabolic properties of PAs can vary substantially and should be taken into account when considering differences in relative potency between different PAs.

Abstract P2-05-07: Pan-cancer analysis of PARP inhibition reveals a suite of biomarkers that correlate with PARP1/2 activity in breast cancer

Abstract The development of multiple PARP inhibitors in BRCA mutant cancers has been one of the most successful implementations of molecularly targeted therapies in oncology. The recent approval of niraparib in BRCA WT Ovarian cancers demonstrates the potential for expansion of PARP inhibitors to other BRCA WT indications. The successful development of PARP inhibitors into new cancer indications will depend on using molecular biomarkers to identify tumors with a unique dependency on PARP1/2 activity. We screened a large (N=523), diverse panel of human cancer cell lines for response to three approved PARP inhibitors: olaparib, niraparib, and talazoparib. Despite differences in relative potency, there was a strong correlation between sensitive and resistant cell lines to all three compounds. In vitro experimentation revealed the inhibitors share a similar biologic mechanism of response, namely a global decrease in PARylation, as well as PARP-trapping at DNA nicks leading to G2 arrest and ultimately cell death. A suite of baseline genomic and proteomic characteristics were found to be strongly associated with response to PARP1/2 inhibition, and were largely consistent between different cancer types. Special attention was paid to BRCA1/2 variants, which represent the only approved patient selection biomarkers. Notably, neither mutations in BRCA1 nor BRCA2 were found to be associated with response to PARP1/2 inhibition in our panel, even when restricting to those with confirmed deleterious ClinVar scores. The molecular predictors of response identified in our screen may ultimately be used to develop diagnostic tools for enrollment into biomarker-enriched clinical trials to expand the use of this promising class of drug into areas of high unmet need. Triple-negative breast cancer represents one of the most intriguing spaces for development. The clinical failures in this space may be partially explained by the lack of empirical predictive biomarkers in the trial design. Our diagnostics may finally allow for the efficacious deployment of PARP inhibitors to triple-negative breast cancer. In particular we have identified a panel of biomarkers that can predict for sensitivity to PARP inhibitors in breast cancer that could be used as patient selection criteria for the expanded clinical development of these compounds into PARP-naïve patient populations with high unmet need. Citation Format: Martina SJ McDermott, Dylan F Conklin, Neil A O'Brien, Kevin Chau, Dennis J Slamon. Pan-cancer analysis of PARP inhibition reveals a suite of biomarkers that correlate with PARP1/2 activity in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-07.

Liver tumor potency indicators for technical toxaphene and congeners simulating weathered toxaphene

ABSTRACTTechnical toxaphene (TT) is a liver tumor promoter in B6C3F1 mice but not in F344 rats. To further evaluate dose-response relationships for weathered toxaphene, B6C3F1 mouse hepatocytes were treated with TT alone, five selected persistent congeners (p-26, p-50, p-62, Hx-Sed, and Hp-Sed), or two selected congener mixtures (simulating weathered toxaphene) and dose-response relationships were characterized for cytotoxicity and gap junction intercellular communication (GJIC) inhibition. Phenobarbital was included as a positive control for mouse liver tumor promotion and GJIC inhibition and dose ranges were calibrated to define benchmark dose concentrations. Each treatment group exhibited significant cytotoxicity and GJIC inhibition for at least one sex (M/F) after 3 and/or 24 h of treatment. Maximum GJIC inhibition was observed at certain noncytotoxic concentrations with sex-specific differences in relative potency estimated as the effective concentration at 20% inhibition (EC20); however, no significant EC20 differences were observed between the treatment groups. Analysis of mixture interactions at the EC20 showed that GJIC inhibition of the two weathered toxaphene mixtures was significantly less than additive compared to that for the component congeners. These findings suggest that the persistent toxaphene congener mixtures tested are not more tumorigenic than the parent insecticide mixture.

Interactions of Cathinone NPS with Human Transporters and Receptors in Transfected Cells.

Pharmacological assays carried out in transfected cells have been very useful for describing the mechanism of action of cathinone new psychoactive substances (NPS). These in vitro characterizations provide fast and reliable information on psychoactive substances soon after they emerge for recreational use. Well-investigated comparator compounds, such as methamphetamine, 3,4-methylenedioxymethamphetamine, cocaine, and lysergic acid diethylamide, should always be included in the characterization to enhance the translation of the in vitro data into clinically useful information. We classified cathinone NPS according to their pharmacology at monoamine transporters and receptors. Cathinone NPS are monoamine uptake inhibitors and most induce transporter-mediated monoamine efflux with weak to no activity at pre- or postsynaptic receptors. Cathinones with a nitrogen-containing pyrrolidine ring emerged as NPS that are extremely potent transporter inhibitors but not monoamine releasers. Cathinones exhibit clinically relevant differences in relative potencies at serotonin vs. dopamine transporters. Additionally, cathinone NPS have more dopaminergic vs. serotonergic properties compared with their non-β-keto amphetamine analogs, suggesting more stimulant and reinforcing properties. In conclusion, in vitro pharmacological assays in heterologous expression systems help to predict the psychoactive and toxicological effects of NPS.

Measured and predicted affinities of binding and relative potencies to activate the AhR of PAHs and their alkylated analogues

Polycyclic aromatic hydrocarbons (PAHs) and their alkylated forms are important components of crude oil. Both groups of PAHs have been reported to cause dioxin-like responses, mediated by aryl hydrocarbon receptor (AhR). Thus, characterization of binding affinity to the AhR of unsubstituted or alkylated PAHs is important to understand the toxicological consequences of oil contamination on ecosystems. We investigated the potencies of major PAHs of crude oil, e.g., chrysene, phenanthrene and dibenzothiophene, and their alkylated forms (n=17) to upregulate expression of AhR-mediated processes by use of the H4IIE-luc transactivation bioassay. In addition, molecular descriptors of different AhR activation potencies among PAHs were investigated by use of computational molecular docking models. Based on responses of the H4IIE-luc in vitro assay, it was shown that potencies of PAHs were determined by alkylation in addition to the number and conformation of rings. Potencies of AhR-mediated processes were generally greater when a chrysene group was substituted, especially in 1-methyl-chrysene. Significant negative correlations were observed between the in vitro dioxin-like potency measured in H4IIE-luc cells and the binding distance estimated from the in silico modeling. The difference in relative potency for AhR activation observed among PAHs and their alkylated forms could be explained by differences among binding distances in the ligand binding domain of the AhR caused by alkylation. The docking model developed in the present study may have utility in predicting risks of environmental contaminants of which toxicities are mediated by AhR binding.

Pityriazepin and other potent AhR ligands isolated from Malassezia furfur yeast

Malassezia furfur yeast strains isolated from diseased human skin preferentially biosynthesize indole alkaloids which can be detected in the human skin and are highly potent activators of the aryl hydrocarbon receptor (AhR) and AhR-dependent gene expression. Chemical analysis of an EtOAc extract of a M. furfur strain obtained from diseased human skin and grown on l-tryptophan agar revealed several known AhR active tryptophan metabolites along with a previously unidentified compound, pityriazepin. While its structure resembled that of the known alkaloid pityriacitrin, the comprised pyridine ring had been transformed into an azepinone. The indoloazepinone scaffold of pityriazepin is extremely rare in nature and has only been reported once previously. Pityriazepin, like the other isolated compounds, was found to be a potent activator of the AhR-dependent reporter gene assay in recombinant cell lines derived from four different species, although significant species differences in relative potency were observed. The ability of pityriazepin to competitively bind to the AhR and directly stimulate AhR DNA binding classified it as a new naturally-occurring potent AhR agonist. M. furfur produces an expanded collection of extremely potent naturally occurring AhR agonists, which produce their biological effects in a species-specific manner.

Species-specific relative AHR1 binding affinities of 2,3,4,7,8-pentachlorodibenzofuran explain avian species differences in its relative potency

Results of recent studies showed that 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are equipotent in domestic chicken (Gallus gallus domesticus) while PeCDF is more potent than TCDD in ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica). To elucidate the mechanism(s) underlying these differences in relative potency of PeCDF among avian species, we tested the hypothesis that this is due to species-specific differential binding affinity of PeCDF to the aryl hydrocarbon receptor 1 (AHR1). Here, we modified a cell-based binding assay that allowed us to measure the binding affinity of dioxin-like compounds (DLCs) to avian AHR1 expressed in COS-7 (fibroblast-like cells). The results of the binding assay show that PeCDF and TCDD bind with equal affinity to chicken AHR1, but PeCDF binds with greater affinity than TCDD to pheasant (3-fold) and Japanese quail (5-fold) AHR1. The current report introduces a COS-7 whole-cell binding assay and provides a mechanistic explanation for differential relative potencies of PeCDF among species of birds.

Cross-species analysis of thyroperoxidase inhibition by xenobiotics demonstrates conservation of response between pig and rat

Thyroperoxidase (TPO), the enzyme that catalyzes the synthesis of thyroid hormone, is a known target for thyroid-disrupting chemicals. In vivo toxicological evidence supporting TPO-inhibition as one molecular-initiating event that leads to thyroid disruption is derived largely from rat models; however, a significant fraction of research on the inhibition of TPO by xenobiotics has been conducted using porcine TPO. The current work tested the hypothesis that porcine and rat thyroid microsomes exposed to TPO-inhibiting chemicals would demonstrate different responses in a guaiacol oxidation assay. A primary objective of this work is to establish the degree of concordance between rat and porcine TPO inhibition data. Microsomes were isolated from both rat and pig thyroid glands, and the guaiacol oxidation assay was performed for a training set of 12 chemicals, including previously reported TPO inhibitors, thyroid-disrupting chemicals thought to perturb other targets, and several previously untested chemicals, to determine the relative TPO inhibition responses across species. Concentration–response curves were derived for methimazole (MMI), dibutylphthalate (DBP), diethylhexylphthalate (DEHP), diethylphthalate (DEP), 3,5-dimethylpyrazole-1-methanol (DPM), iopanoic acid (IOA), 2-mercaptobenzothiazole (MBT), sodium perchlorate (PERC), p-nonylphenol (PNP), 4-propoxyphenol (4POP), 6-propylthiouracil (PTU), and triclosan (TCS). MMI, PTU, MBT, DPM, 4POP, and at extremely high concentrations, PERC, inhibited TPO activity. Results demonstrated a strong qualitative concordance of response between the two species. All chemicals that inhibited TPO in porcine microsomes also inhibited TPO in rat microsomes. Hill model-derived IC50 values revealed approximate 1.5- to 50-fold differences in relative potency to MMI between species for positive chemicals. DPM, MBT, 4POP, and PTU exhibited greater relative potency to MMI using rat TPO versus porcine TPO, but rank order potency for inhibition was similar for the other test chemicals, with: PTU>MBT>DPM>4POP>PERC for rat TPO and MBT>PTU>DPM>4POP>PERC for porcine TPO. These data support the extrapolation of porcine TPO data to potential thyroid-disrupting activity in rodent models to evaluate TPO-inhibiting chemicals.

The biological assay of Rauwolfia schueli.

Abstract The biological effects of Rauwolfia schueli are essentially the same as those of the pure alkaloid reserpine and the crude compound R. serpentina in the rat. No difference in relative potency between the small and large roots of large trees of the R. schueli species was apparent when either reserpine or R. serpentina was employed as the standard. R. schueli was estimated to be about 1/630 as active as reserpine. In terms of reserpine-like activity, this species appeared to be more potent than R. serpentina and by comparison with the results of others about as potent as R. canescens but more potent than R. heterophylla. Within the range of doses selected, blood pressure did not appear to be an adequate index for measuring reserpine-like activity in the rat.

In Vitro Toxicity Profiling of Ultrapure Non–Dioxin-like Polychlorinated Biphenyl Congeners and Their Relative Toxic Contribution to PCB Mixtures in Humans

The toxic equivalency concept used for the risk assessment of polychlorinated biphenyls (PCBs) is based on the aryl hydrocarbon receptor (AhR)-mediated toxicity of coplanar dioxin-like (DL) PCBs. Most PCBs in the environment, however, are non-dioxin-like (NDL) PCBs that cannot adopt a coplanar structure required for AhR activation. For NDL-PCBs, no generally accepted risk concept is available because their toxicity is insufficiently characterized. Here, we systematically determined in vitro toxicity profiles for 24 PCBs regarding 10 different mechanisms of action. Prior to testing, NDL-PCB standards were purified to remove traces of DL compounds. All NDL-PCBs antagonized androgen receptor activation and inhibited gap junctional intercellular communication (GJIC). Lower chlorinated NDL-PCBs were weak estrogen receptor (ER) agonists, whereas higher chlorinated NDL-PCBs were weak ER antagonists. Several NDL-PCBs inhibited estradiol-sulfotransferase activity and bound to transthyretin (TTR) but with much weaker potencies than reported for hydroxylated PCB metabolites. AhR-mediated expression of uridine-glucuronyl transferase isozyme UGT1A6 was induced by DL-PCBs only. Hierarchical cluster analysis of the toxicity profiles yielded three separate clusters of NDL-PCBs and a fourth cluster of reference DL-PCBs. Due to small differences in relative potency among congeners, the highly abundant indicator PCBs 28, 52, 101, 118, 138, 153, and 180 also contributed most to the antiandrogenic, (anti)estrogenic, antithyroidal, tumor-promoting, and neurotoxic potencies calculated for PCB mixtures reported in human samples, whereas the most potent AhR-activating DL-PCB-126 contributed at maximum 0.2% to any of these calculated potencies. PCB-168 is recommended as an additional indicator congener, given its relatively high abundance and antiandrogenic, TTR-binding, and GJIC-inhibiting potencies.

Analysis of the CYP1A1 mRNA Dose-Response in Human Keratinocytes Indicates that Relative Potencies of Dioxins, Furans, and PCBs Are Species and Congener Specific

Reports indicate that toxic equivalency factors (TEFs) based primarily on rodent data do not accurately predict in vitro human responsiveness to certain dioxin-like chemicals (DLCs). To investigate this in cells responsive to dioxins and relevant to chloracne, normal human epidermal keratinocytes were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and several DLCs, each with a TEF value of 0.1, representing three classes of congeners. We estimated half maximal effective concentration (EC50)-based donor-specific relative potency (REP) values for cytochrome P450 1A1 (CYP1A1) messenger RNA (mRNA) induction for TCDD, 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,6,7,8-hexachlorodibenzofuran (HxCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126). We also determined EC50-based population-level REP values (n = 4) for CYP1A1 mRNA induction for TCDD, HxCDF, and PCB 126. Furthermore, an alternative factor, the relative threshold factor (RTF) based on the low end (threshold) of the dose-response curve, was calculated. Our results demonstrated that HxCDF had a population-based REP value of 0.98, 9.8-fold higher than its assigned TEF value of 0.1. Conversely, PCB 126 had an REP value of 0.0027 and an RTF of 0.0022, 37-fold and 45-fold less than its assigned TEF of 0.1, respectively. The REP values for HxCDD and TCDF were 0.24 and 0.10, respectively, similar to their assigned value of 0.1. Therefore, although the DLCs tested in the current study all possessed the same assigned TEF value of 0.1, congener-specific differences in REPs and RTFs were observed for human keratinocytes. These congener-specific discrepancies are likely because of differences in interspecies factors that have yet to be defined.

Opioids for chronic noncancer pain: a new Canadian practice guideline.

Six allosteric HIV-1 entry inhibitor modulators of the chemokine (C-C motif) receptor 5 (CCR5) receptor are compared for their potency as inhibitors of HIV-1 entry [infection of human osteosarcoma (HOS) cells and peripheral blood mononuclear cells (PBMC)] and antagonists of chemokine (C-C motif) ligand 3-like 1 [CCL3L1]-mediated internalization of CCR5. This latter activity has been identified as a beneficial action of CCL3L1 in prolonging survival after HIV-1 infection ( Science307<b>:</b>1434-1440, 2005 ). The allosteric nature of these modulators was further confirmed with the finding of a 58-fold (HOS cells) and 282-fold (PBMC) difference in relative potency for blockade of CCL3L1-mediated internalization versus HIV-1 entry. For the CCR5 modulators, statistically significant differences in this ratio were found for maraviroc, vicriviroc, aplaviroc, Sch-C, TAK652, and TAK779. For instance, although TAK652 is 13-fold more potent as an HIV-1 inhibitor (over blockade of CCL3L1-mediated CCR5 internalization), this ratio of potency is reversed for Sch-C (22-fold more potent for CCR5-mediated internalization over HIV-1 entry). Quantitative analyses of the insurmountable antagonism of CCR5 internalization by these ligands suggest that all of them reduce the efficacy of CCL3L1 for CCR5 internalization. The relatively small magnitude of dextral displacement accompanying the depression of maximal responses for aplaviroc, maraviroc and vicriviroc suggests that these modulators have minimal effects on CCL3L1 affinity, although possible receptor reserve effects obscure complete interpretation of this effect. These data are discussed in terms of the possible benefits of sparing natural CCR5 chemokine function in HIV-1 entry inhibition treatment for AIDS involving allosteric inhibitors.

The Relative Activity of “Function Sparing” HIV-1 Entry Inhibitors on Viral Entry and CCR5 Internalization: Is Allosteric Functional Selectivity a Valuable Therapeutic Property?

Six allosteric HIV-1 entry inhibitor modulators of the chemokine (C-C motif) receptor 5 (CCR5) receptor are compared for their potency as inhibitors of HIV-1 entry [infection of human osteosarcoma (HOS) cells and peripheral blood mononuclear cells (PBMC)] and antagonists of chemokine (C-C motif) ligand 3-like 1 [CCL3L1]-mediated internalization of CCR5. This latter activity has been identified as a beneficial action of CCL3L1 in prolonging survival after HIV-1 infection ( Science 307: 1434-1440, 2005 ). The allosteric nature of these modulators was further confirmed with the finding of a 58-fold (HOS cells) and 282-fold (PBMC) difference in relative potency for blockade of CCL3L1-mediated internalization versus HIV-1 entry. For the CCR5 modulators, statistically significant differences in this ratio were found for maraviroc, vicriviroc, aplaviroc, Sch-C, TAK652, and TAK779. For instance, although TAK652 is 13-fold more potent as an HIV-1 inhibitor (over blockade of CCL3L1-mediated CCR5 internalization), this ratio of potency is reversed for Sch-C (22-fold more potent for CCR5-mediated internalization over HIV-1 entry). Quantitative analyses of the insurmountable antagonism of CCR5 internalization by these ligands suggest that all of them reduce the efficacy of CCL3L1 for CCR5 internalization. The relatively small magnitude of dextral displacement accompanying the depression of maximal responses for aplaviroc, maraviroc and vicriviroc suggests that these modulators have minimal effects on CCL3L1 affinity, although possible receptor reserve effects obscure complete interpretation of this effect. These data are discussed in terms of the possible benefits of sparing natural CCR5 chemokine function in HIV-1 entry inhibition treatment for AIDS involving allosteric inhibitors.

Agreement between two methods for assessing bioequivalence of inhaled salbutamol

For inhaled medications, bioequivalence testing is becoming increasingly important owing to the availability of many inhalation devices. We evaluated agreement between the Finney bioassay and the E max model in the assessment of bioequivalence of salbutamol administered via a metered-dose inhaler with a spacer (pMDI+Volumatic) or via a dry-powder inhaler (Diskus) in asthmatic patients with methacholine-induced bronchoconstriction. Eighteen patients inhaled methacholine until FEV 1 decreased by ≈35% of control. Following inhalation of placebo, 200 and 400 mcg salbutamol through the pMDI+Volumatic or the Diskus, changes in FEV 1 were repeatedly measured over a 60-min observation period. Bioequivalence of salbutamol administered via the two inhalation devices was assessed by calculating the relative potency of each device by using the Finney 2-by-2 parallel regression analysis and the non-linear, E max model. Agreement between these methods in calculating relative potency was evaluated by using the Bland–Altman method. After salbutamol FEV 1 values were similar irrespective of the device employed, and greater ( P<0.01) than those after placebo. However, assessment of relative potencies obtained with both the Finney (1.97, 90% CI 1.62–2.32) and the E max (2.25, 90% CI 1.90–2.60) methods revealed that twice the salbutamol dose was needed to reverse methacholine-induced bronchoconstriction when the drug was inhaled via the Diskus than via the pMDI+Volumatic. The mean difference in relative potency (−0.28, 90% CI −0.001 to −0.56) calculated with both methods did not significantly differ from zero, and none of the individual differences exceeded the limits of agreement. The Finney and the E max methods provide comparable results in the evaluation of bioequivalence of different salbutamol formulations. The pMDI+Volumatic is twice as efficient as the Diskus in lung delivery of salbutamol in asthma patients.

Structure-Toxicity Analysis of Type-2 Alkenes: In Vitro Neurotoxicity

Acrylamide (ACR) is a conjugated type-2 alkene that produces synaptic toxicity presumably by sulfhydryl adduction. The alpha,beta-unsaturated carbonyl of ACR is a soft electrophile and, therefore, adduction of nucleophilic thiol groups could occur through a conjugate (Michael) addition reaction. To address the mechanism of thiol adduct formation and corresponding neurotoxicological importance, we defined structure-toxicity relationships among a series of conjugated type-2 alkenes (1 microM-10mM), which included acrolein and methylvinyl ketone. Results show that exposure of rat striatal synaptosomes to these chemicals produced parallel, concentration-dependent neurotoxic effects that were correlated to loss of free sulfhydryl groups. Although differences in relative potency were evident, all conjugated analogs tested were equiefficacious with respect to maximal neurotoxicity achieved. In contrast, nonconjugated alkene or aldehyde congeners did not cause synaptosomal dysfunction or sulfhydryl loss. Acrolein and other alpha,beta-unsaturated carbonyls are bifunctional (electrophilic reactivity at the C-1 and C-3 positions) and could produce in vitro neurotoxicity by forming protein cross-links rather than thiol monoadducts. Immunoblot analysis detected slower migrating, presumably derivatized, synaptosomal proteins only at very high acrolein concentrations (>or= 25 mM). Exposure of synaptosomes to high concentrations of ACR (1M), N-ethylmaleimide (10mM), and methyl vinyl ketone (MVK) (100mM) did not alter the gel migration of synaptosomal proteins. Furthermore, hydralazine (1mM), which blocks the formation of protein cross-links, did not affect in vitro acrolein neurotoxicity. Thus, type-2-conjugated alkenes produced synaptosomal toxicity that was linked to a loss of thiol content. This is consistent with our hypothesis that the mechanism of ACR neurotoxicity involves formation of Michael adducts with protein sulfhydryl groups.

Uncertainties Associated with Assessing the Risk of an Endocrine Active Substance in the Canadian Environment

Abstract A number of biological responses and multigenerational effects, mediated through the disruption of endocrine systems, have been observed in biota exposed to relatively low concentrations of environmental contaminants. These types of responses need to be considered within a weight of evidence approach in our risk assessment and risk management frameworks. However, including endocrine responses in an environmental risk assessment introduces a number of uncertainties that must be considered. A risk assessment of nonylphenol and nonylphenol polyethoxylates (NP/NPE) is used as a case study to demonstrate the sources and magnitude of some of the uncertainties associated with using endocrine disruption as an assessment endpoint. Even with this relatively well studied group of substances, there are substantial knowledge gaps which contribute to the overall uncertainties, limiting the interpretation within the risk assessment. The uncertainty of extrapolating from in vitro or biochemical responses to higher levels of organization or across species is not well understood. The endocrine system is very complex and chemicals can interact or interfere with the normal function of endocrine systems in a number of ways (e.g., receptors, hormones) which may or may not result in an adverse responses in the whole organism. Using endocrine responses can lead to different conclusions than traditional endpoints due to a variety of factors, such as differences in relative potencies of chemicals for specific endpoints (e.g., receptor binding versus chronic toxicity). The uncertainties can also be considerably larger and the desirability of using endocrine endpoints should be carefully evaluated. Endocrine disruption is a mode of action and not a functional endpoint and this needs to be considered carefully in the problem formulation stage and the interpretation of the weight of evidence.

Potency, Impotency, and Importance!

Potency, Impotency, and Importance!

Transactivation Activity of Human, Zebrafish, and Rainbow Trout Aryl Hydrocarbon Receptors Expressed in COS-7 Cells: Greater Insight into Species Differences in Toxic Potency of Polychlorinated Dibenzo-p-dioxin, Dibenzofuran, and Biphenyl Congeners

Transactivation assays were used to compare the potency and efficacy of polychlorinated dibenzo-p-dioxin (PCDD), dibenzofuran (PCDF), and biphenyl (PCB) congeners in activating aryl hydrocarbon receptors (AhRs) from rainbow trout (rtAhR2α and rtAhR2β), zebrafish (zfAhR2), and human (huAhR), respectively. All AhRs were expressed with their species-specific AhR nuclear translocator (ARNT) in COS-7 cells. Transactivation activity was determined for two PCDD, two PCDF, and seven PCB congeners with each of the four AhR/ARNT pairs using prt1Aluc, a luciferase reporter driven by two dioxin-responsive enhancer elements (DREs) from the rainbow trout cyp1A gene. Maximal-fold induction, EC50, and relative potency values were calculated for congeners that exhibited dose-related activity in the assay. Of the four AhR/ARNT pairs tested with PCDD, PCDF, and non-ortho PCB congeners, three exhibited high activity (rainbow trout AhR2α, zebrafish AhR2, and human AhR), while rainbow trout AhR2β had very weak or no activity. Comparisons between these AhRs showed that while mono-ortho PCBs were able to activate the human AhR, they were generally ineffective in activating rainbow trout and zebrafish AhR2s. This supports the hypothesis that structural differences between mammalian and fish AhRs may account for differences in relative potencies of the mono-ortho PCBs between mammals and fish. Another important finding was a significant difference in transactivation activity between the two rainbow trout AhR2 isoforms despite the fact that they are 95% identical at the amino acid level. For all PCDD, PCDF, and PCB agonists tested, rainbow trout AhR2α was significantly more active than AhR2β. However, rainbow trout AhR2β is active as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-activated transcription factor, with enhancer elements from the mouse cyp1A gene. This suggests that AhR2β may have evolved to serve a different physiological function than AhR2α in salmonid fish species.

Betidamino acid scan of the GnRH antagonist acyline.

Strong clinical evidence suggests that GnRH antagonists will replace GnRH agonists in a number of indications because of their ability to inhibit gonadotropin secretion as long as an adequate concentration of the analogue is present in the circulation whereas superagonists will take approximately 2 weeks to desensitize the gonadotrophs. Until recently, antagonists were either too weak and/or would release histamine. Azaline B {[Ac-D2Nal1,D4Cpa2,D3Pal3, 4Aph5(atz),D4Aph6(atz),ILys8,DAla10] GnRH} and long-acting members of the azaline family {Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(X)-D4Aph(Y) -Leu-ILys-Pro-DAla-NH2}, however, appear to be promising drug candidates. Because these antagonists tend to form gels (due to the formation of beta-sheet structures) and, as a result, are not readily amenable to formulation for long-term delivery, we have investigated ways of increasing hydrophilicity while retaining high potency and lack of histamine releasing activity. Betidamino acids (a contraction of "beta" position and "amide") are N'-monoacylated (optionally, N'-monoacylated and N-mono- or N,N'-dialkylated) aminoglycine derivatives in which each N'-acyl/alkyl group may mimic naturally occurring amino acid side chains or introduce novel functionalities. We have used unresolved N alpha-Boc,N'alpha-Fmoc-aminoglycine, and N alpha-Boc,N'alpha-(CH3)Fmoc-aminoglycine as templates for the introduction of betidamino acids in acyline (Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(A c)-Leu-Ilys-Pro-DAla-NH2), a long acting member of the azaline B family, to test biocompatibility of these betide derivatives. Diastereomeric peptides could be separated using RP-HPLC in most cases. Biological results obtained in vitro (binding affinity to rat pituitary gland membranes) and in vivo (rat antiovulatory assay, AOA) indicate in most cases small differences in relative potencies (< 5-fold) between the D- and L-nonalkylated betidamino acid-containing acylines. Importantly, most betide diastereomers have high affinity for the GnRH receptor and were equipotent with acyline in the AOA. Greater differences in affinity and potency between diastereomers were observed after introduction of a methyl group on the side chain nitrogen ("beta" position) of the same analogues, with one of the diastereomer having an affinity and a potency in the AOA equivalent to that of acyline. These results suggest that chirality at the alpha-carbon coupled to side chain orientation is important for receptor recognition. The duration of action of some of the most potent analogues was also determined in the castrated male rat in order to measure the extent (efficacy and duration of action) of inhibition of luteinizing hormone release. Data suggest that introduction of a betidamino acid results in reduction of duration of action. Also, introduction of betidamino acids results in peptides with increased hydrophilicity (as determined by elution times on C18 silicas at pH 7.3) compared to that of the parent compound. N'-Methyl substitution results in parallel increase in retention times on C18 silicas as expected.

Effects of 17β-estradiol and 4-nonylphenol on smoltification and vitellogenesis in Atlantic salmon (Salmo salar)

The impact of 17β-estradiol (E2) and the putative estrogenic compound, 4-nonylphenol (4-NP), on smoltification and vitellogenesis in Atlantic salmon (Salmo salar) was investigated during a 30 day period starting late April. Three groups of fresh water (FW) fish (1 year old, mixed sexes, average weight 23 g) were injected once a week with 50 µg (0.18 µmol) 17β-estradiol, 3 mg (13.6 µmol) 4-nonylphenol dissolved in peanut oil, or peanut oil alone as control. Every ten days, subgroups were challenged with 28 ppt seawater (SW) for 24h, and sampled together with subgroups of FW fish. Treatment effects were examined on vitellogenic and osmoregulatory parameters. E2 and 4-NP treatment increased the total calcium and protein level in plasma and the hepatosomatic index of FW fish, both indicating an activated vitellogenesis in the liver. The presence of vitellogenin in the plasma of 4-NP- and E2-treated groups was further indicated by the appearance of a high molecular weight vitellogenin band (550 kDa) in electropherograms produced by native gel electrophoresis. This band appeared in exactly the same position in both the E2- and the 4-NP-treated groups but could not be detected in controls. During the 30 day treatment period, control fish approached the peak of smoltification, as indicated by a distinct silvery appearance, decreasing condition factor, increasing levels of gill Na+,K+-ATPase and improved hypoosmoregulatory performance in the SW-challenge test. Both E2 and 4-NP treatments significantly inhibited the progress of smoltification, as judged by a significant reduction of gill Na+,K+-ATPase activity, relative α-subunit Na+,K+-ATPase mRNA expression, gill chloride cell density and a poorer hypoosmoregulatory performance of treated fish. The impaired SW-tolerance of E2- and 4-NP-treated fish was strongly correlated with a decreased gill Na+,K+-ATPase activity. Despite a difference in relative potency, the present study shows that 4-nonylphenol and 17β-estradiol may have qualitatively similar inhibitory effects on smoltification and hypoosmoregulatory physiology of Atlantic salmon. Both 4-NP and E2 activated the vitellogenic system, and the study supports the hypothesis that sexual maturation and smoltification are antagonistic, developmental phenomena in salmon. It is suggested that the presence of estrogenic compounds in the environment may negatively influence smoltification and migration in wild stocks of salmon.