Potent Congener Research Articles

Selection of human immunodeficiency virus type 1 resistance against the pyranodipyrimidine V-165 points to a multimodal mechanism of action

We have previously identified the pyranodipyrimidines (PDPs) as a new class of integrase (IN) inhibitors. The most potent congener V-165 inhibits HIV-1 integration at low micromolar concentrations by inhibiting the binding of IN to the DNA. As part of pre-clinical studies with PDP, we wanted to investigate HIV resistance development against V-165 and to further characterize the physicochemical properties of the compound. We selected PDP-resistant HIV-1 strains by growing the virus in the presence of increasing concentrations of V-165. The selected strains were analysed genotypically and phenotypically. Mutant IN enzymes were generated and evaluated in an enzymatic oligonucleotide-based assay for their activity and sensitivity to the different IN inhibitors. In addition, the antiviral effect of the compound on viral entry and integration was measured using quantitative PCR. Numerous mutations were detected in the RT, IN and env genes of the virus selected in the presence of V-165. Although V-165 inhibited integration in vivo as indicated by a decrease in the number of integrated proviruses, the compound also inhibited viral entry at a concentration of 19 microM. V-165 was poorly recovered from human hepatic microsomal matrix and 1% BSA. These data point to a multimodal mechanism of action. A quest for derivatives of V-165 that specifically target IN should be pursued.

Differences in acute toxicity syndromes of 2,3,7,8-tetrachlorodibenzo- p-dioxin and 1,2,3,4,7,8-hexachlorodibenzo- p-dioxin in rats

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is the most potent congener of polychlorinated dibenzo- p-dioxins. The potency of 1,2,3,4,7,8-hexachlorodibenzo- p-dioxin (HxCDD) is only 10% of that of TCDD for typical aryl hydrocarbon receptor (AHR)-mediated effects. Acute lethality, macroscopic effects, and liver toxicity of TCDD and HxCDD were compared in male rats of the strain Han/Wistar ( Kuopio; H/W), and of the lines A and B. The latter two rat lines originate from crossbreeding of H/W and Long-Evans ( Turku/ AB) rats. H/W and line A rats are highly resistant to acute toxicity of TCDD due to an altered AHR, while line B rats are moderately resistant due to H/W-type alleles of another, yet unidentified gene contributing to TCDD resistance (“gene B”). The rats received 200–10,000 μg/kg of either TCDD or HxCDD intragastrically and were monitored for 46 days. In all rats, the highest dose of HxCDD (10,000 μg/kg) reduced body weight more effectively than an identical dose of TCDD. Only HxCDD (10,000 μg/kg) caused gastrointestinal hemorrhage, pale (fatty) livers and death by day 15 in H/W and line A rats. In line B rats, HxCDD caused pronounced hepatic fatty degeneration, whereas TCDD induced hepatic accumulation of biliverdin and its derivatives. Both congeners induced sinusoidal distension in liver. In H/W and line A rats, the estimated LD 50 values were >10,000 μg/kg and 2000–10,000 μg/kg for TCDD and HxCDD, respectively; for line B rats they were 480 μg/kg and 1000–2000 μg/kg, respectively. Thus, HxCDD was more potent than TCDD in inducing acute mortality in H/W and line A rats, contrary to what is predicted by toxic equivalency factor (TEF) values. In line B, the expected rank order of potencies prevailed. These findings suggest that in addition to the canonical AHR-mediated toxic pathways, HxCDD possesses an AHR-independent mechanism of toxicity, whose main manifestations are rapid body weight loss, mortality, fatty liver and gastrointestinal hemorrhage.

Fractionation and Determination of Ah Receptor (AhR) Agonists in Organic Waste After Anaerobic Biodegradation and in Batch Experiments with PCB and decaBDE (8 pp)

Anaerobic digestion of organic household waste can lead to an increase in dioxin-like content, as determined by dioxin-specific bioassays. This may be a result of bioactivation of Ah receptor (AhR) agonists into more potent congeners. Work towards identifying the contributing compound groups is important in order to understand the mechanisms and to assess the relevance behind this increase in dioxin-like toxicity, since the residue can be used as a soil fertilising agent. The aim with the present work was to identify compound groups with AhR agonistic properties that caused the previously reported increase in dioxin-like activity after anaerobic biodegradation Firstly, chemical fractionation combined with dioxin bioassay testing was used to find bioactive classes of compounds. Secondly, batch digestion experiments with an externally added polychlorinated biphenyl (PCB) mixture (Clophen A50) and with decabrominated diphenyl ether (decaBDE), respectively, were studied as a possible process for transformation of precursors into more potent, dioxin-like compounds. Mesophilic (37ºC) and thermophilic (55ºC) anaerobic digestion were studied. Two different dioxin-specific bioassays were used to analyse AhR agonists in the biodegraded material, the CELCAD and the DR-CALUX. AhR agonist activity was detected in both di- and polyaromatic fractions of digestate extracts, which indicated that a diverse mixture of compounds contributed to the bioassay responses. No quantifiable activities were induced by the monoaromatic fractions. Further fractionation based on planarity revealed higher concentrations of AhR agonists than what was detected after the first fractionation, probably due to non-additive biological interactions of compounds in the extract that were removed in the second fractionation. These results showed significant activity in the non-planar diaromatic fractions and in the co-planar fractions of both diaromates and polyaromates. In the batch experiment with externally added PCB, an increase in dioxin-like activity was seen after 21 days of digestion at mesophilic conditions. After completed digestion, the content of AhR agonists was equal to the start concentration. PCB analysis with GC-MS indicated that dehalogenation of PCBs occurred in the digestors. The batch experiment with decaBDE showed no significant changes in TEQ-concentrations over time. The results show that the previously reported increase of AhR agonists during mesophilic anaerobic digestion is probably due to an accumulation of several different groups of AhR agonists, both diaromatic and polyaromatic, and both co-planar and non-planar. Batch experiments with externally added PCBs and decaBDE, respectively, did not result in any accumulation of AhR agonist activity after completed digestion, even though chemical analysis indicate a dechlorination of PCBs. Complex, unfractionated extracts were difficult to test using the bioassay approach. Removal of AhR antagonists or otherwise interacting compounds during fractionation may yield bio-TEQ values that are much higher than in the original extract. . Our results indicate that the environmental risk that AhR agonists may pose concerning large-scale anaerobic digestion of organic household waste probably depends on the efficiency of the digester and the sludge residence time. In order to obtain reliable results with the bioassays, an extensive cleanup and fractionation procedure is necessary. Without clean up and fractionation, there is a risk for false negatives and misleading conclusions. DR-CALUX and CELCAD were both suitable for these kinds of studies, provided that suitable fractionation methods are used.

Uncertainties related to the assignment of a toxic equivalency factor for 1,2,3,4,6,7,8,9-octachlorodibenzo- p-dioxin

The Toxic Equivalency Factor (TEF) approach is a methodology that assigns relative toxicity values to structurally related chemicals in comparison to a reference chemical. For the polychlorinated dibenzo- p-dioxins (PCDDs), the reference is the most potent congener, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Here, we critically review the literature on the effects of a weak PCDD, 1,2,3,4,6,7,8,9-octachlorodibenzo- p-dioxin (OCDD), and describe the uncertainties of assigning its TEF. PCDDs, including OCDD, are less potent in human cell models compared to the rat models from which the TEF are estimated. This lack of concordance is even more pronounced with the weaker congeners such as OCDD. Furthermore, OCDD is also likely to compete with TCDD for binding to cytochrome P4501A2 (CYP1A2), effectively decreasing the hepatic tissue/fat ratio of TCDD. Overall, the predictive value of TEFs would be improved by incorporating into this number the relative sensitivity of human cell responses compared to rodent responses, by determining the toxicological effects of altering the tissue distribution of dioxin-like compounds through competition for CYP-binding sites, and by understanding the mechanism of cancer causation of any dioxin and whether this mechanism is conserved in humans and at equivalent doses.

Structure-Based Design, Parallel Synthesis, Structure−Activity Relationship, and Molecular Modeling Studies of Thiocarbamates, New Potent Non-Nucleoside HIV-1 Reverse Transcriptase Inhibitor Isosteres of Phenethylthiazolylthiourea Derivatives

In this paper we describe our structure-based ligand design, synthetic strategy, and structure-activity relationship (SAR) studies that led to the identification of thiocarbamates (TCs), a novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), isosteres of phenethylthiazolylthiourea (PETT) derivatives. Assuming as a lead compound O-[2-(phthalimido)ethyl]phenylthiocarbamate 12, one of the precursors of the previously described acylthiocarbamates (Ranise, A.; et al. J. Med. Chem. 2003, 46, 768-781), two targeted solution-phase TC libraries were prepared by parallel synthesis. The lead optimization strategy led to para-substituted TCs 31, 33, 34, 39, 40, 41, 44, 45, and 50, which were active against wild-type HIV-1 in MT-4-based assays at nanomolar concentrations (EC50 range: 0.04-0.01 microM). The most potent congener 50 (EC50 = 0.01 microM) bears a methyl group at position 4 of the phthalimide moiety and a nitro group at the para position of the N-phenyl ring. Most of the TCs showed good selectivity indices, since no cytotoxic effect was detected at concentrations as high as 100 microM. TCs 31, 37, 39, 40, and 44 significantly reduced the multiplication of the Y181C mutant, but they were inactive against K103R and K103N + Y181C mutants. Nevertheless, the fold increase in resistance of 41 was not greater than that of efavirenz against the K103R mutant in enzyme assays. The docking model predictions were consistent with in vitro biological assays of the anti-HIV-1 activity of the TCs and related compounds synthesized.

PHOTOLABELING PROBES OF RIBAVIRIN AND EICAR

Ribavirin, the only small molecule available so far for treating hepatitis C virus infection, was recently used in an emergency context to treat patients with severe acute respiratory syndrome in the early stages of the disease. EICAR, one of the most potent congeners of ribavirin, has 10 to 100 times greater antiviral potency than ribavirin. The mechanisms underlying the antiviral effects of ribavirin and EICAR have not yet been definitely elucidated, but they seem to be similar. In order to study the mechanisms responsible for their antiviral effects using a photolabeling approach, we have developed photolabeling probes of ribavirin and EICAR, in which an azido group was introduced into the pseudobases of triazole and imidazole, respectively. The ribavirin photoprobes were obtained by directly coupling the azidotriazole to the protected ribose sugar, while the EICAR probe was prepared by diazotizing AICAR and subsequently substituting with NaN3. All these probes showed a fast, clear-cut photochemical reaction, which suggests that they are promising tools for use in photolabeling studies.

Optically active mexiletine analogues as stereoselective blockers of voltage-gated Na(+) channels.

Optically active mexiletine analogues were synthesized and evaluated in vitro as use-dependent blockers of skeletal muscle sodium channels. The mexiletine analogues were obtained by replacing either the methyl group on the stereogenic center of mexiletine [1-(2,6-dimethylphenoxy)propan-2-amine] with a phenyl group or modifying the phenoxy moiety (by removal of one or both of the methyl groups, or introducing a chlorine atom), or both. The voltage clamp recordings showed that, regardless of the substitution pattern of the aryloxy moiety, all the compounds bearing a phenyl group on the stereogenic center (3a-f) were more active than mexiletine both in tonic and phasic block. This observation was in contrast with what was observed for mexiletine, where the removal of both methyls from the aryloxy moiety caused a dramatic reduction of potency. The most potent congener, (R)-2-(2-methylphenoxy)-1-phenylethanamine [(R)-3b], was 27-fold more potent than (R)-mexiletine in producing a tonic block, i.e., the reduction of peak sodium current in resting conditions after application of the compound. (R)-3b maintained a use-dependent behavior, being 23-fold more potent in condition of high frequency of stimulation (phasic block). Despite what was observed with mexiletine, the stereoselectivity held in phasic block conditions. Stereoselectivity indexes were generally low, ranging from 1 to 4, but except for that of the 2,6-xylyloxy congener 3c, they were higher for the congeners bearing a phenyl ring on the stereogenic center than for mexiletine and its strictly related analogue 1-methyl-2-phenoxyethanamine (1). This finding was in agreement with Pfeiffer's rule. The introduction of a chlorine atom in the 4-position of the aryloxy moiety caused a reduction of potency and a reversal of stereoselectivity as well. On the basis of the model to date accepted for the sodium channel local anesthetic-like molecule receptor, some possible explanations of our observations will be proposed.

The effect of remote chirality on the antibacterial activity of indolinyl, tetrahydroquinolyl and dihydrobenzoxazinyl oxazolidinones

The oxazolidinones are promising agents for the treatment of infections caused by gram-positive bacteria, including multidrug-resistant strains. In ongoing studies we have discovered that a strategically placed chiral center of appropriate absolute configuration improves the antibacterial activity of indolinyl oxazolidinone analogues (gram-positive MIC's<0.5 μg/mL for the most potent congeners). The design, synthesis, antibacterial activity and pharmacokinetic profile of a selected series of α-methylated indoline derivatives and a related set of tetrahydroquinolyl and dihydrobenzoxazinyl analogues are discussed.

New Class of HIV Integrase Inhibitors that Block Viral Replication in Cell Culture

Background: To improve the existing combination therapies of infection with the human immunodeficiency virus (HIV) and to cope with virus strains that are resistant to multiple drugs, we initiated a search for effective inhibitors of HIV integrase, the enzyme responsible for inserting the viral cDNA into the host cell chromosome.Results: We have now identified a series of 5H-pyrano[2,3-d:-6,5-d′]dipyrimidines that block the replication of various strains of HIV-1 and HIV-2. The most potent congener, 5-(4-nitrophenyl)-2,8-dithiol-4,6-dihydroxy-5H-pyrano[2,3-d:-6,5-d′]dipyrimidine (V-165), inhibited the replication of HIV-1(IIIB) in MT-4 cells at a 50% effective concentration (EC50) of 8.9 μM, which is 14-fold below its cytotoxic concentration. V-165 was equally active against virus strains that were resistant toward inhibitors of viral entry or reverse transcriptase. In combination regimens in cell culture, V-165 acted subsynergistically with zidovudine or nelfinavir and synergistically with nevirapine. V-165 inhibited both reverse transcriptase and integrase activities in enzymatic assays at micromolar concentrations, but only a close correlation was found between the anti-HIV activity observed in cell culture and the inhibitory activity in the integrase strand transfer assays. Time-of-addition experiments indicated that V-165 interfered with the viral replication cycle at a time point coinciding with integration. Quantitative Alu-PCR corroborated that the anti-HIV activity of V-165 is based upon the inhibition of proviral DNA integration.Conclusions: Based on their mode of action, which is different from that of clinically approved anti-HIV drugs, PDPs are good candidates for further development into new drugs and to be included in future combination regimens.

Structure–Activity Relationships and Dose Responses of Polychlorinated Dibenzo-p-dioxins for Short-Term Effects in 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Resistant and -Sensitive Rat Strains

Dose responses of the characteristic short-term effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD), and 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) were compared in the resistant Han/Wistar (Kuopio) (H/W) rats and the sensitive Long-Evans (Turku/AB) (L-E) rats. The resistance of H/W rats is linked to the altered H/W-type aryl hydrocarbon receptor (AHR). Exceptionally, in terms of acute lethality, the most potent congener for H/W rats is HxCDD, followed by HpCDD, PeCDD, and TCDD. The study objectives were to find out if this exceptional sensitivity of H/W rats also holds for nonlethal toxic endpoints and to compare potency and efficacy (magnitude of effect) of PCDDs between L-E and H/W rats. Dose responses for several endpoints were determined, modeled, and used for ED50 and relative potency (REP) calculations. For all endpoints measured, TCDD was the most potent congener, followed by PeCDD, HxCDD, and HpCDD in both strains, and the REP estimates were consistent with the current toxic equivalency factors (TEFs). For most endpoints, H/W rats showed smaller responses to all congeners than L-E rats, and this difference was due to lower efficacy rather than lower potency. H/W rats showed lower efficacy to body weight loss, serum aspartate aminotransferase activity, and serum concentrations of total bilirubin, free fatty acids, and thyroxine. In contrast, effects on cytochrome P4501A1 induction, thymus atrophy, and dental defects were similar in both strains. In conclusion, the results are in agreement with the current WHO-TEFs and imply that relative potency values derived from mortality are not necessarily valid for other endpoints. The results support our previous observations about two different types of AHR-mediated mechanisms. Type I effects are similar in both strains, and type II effects show decreased efficacy of toxic response in relation with the altered H/W-type AHR.

Phospholipase A2-Mediated Ca2+ Influx by 2,2′,4,6-Tetrachlorobiphenyl in PC12 Cells

Polychlorinated biphenyls (PCBs) are a group of persistent and widespread environmental pollutants, and known to affect signaling molecules. Phospholipase A2 (PLA2) mediates cellular destructive processes as well as normal physiological responses in neuronal cells. In this study, we examined whether PLA2 can be activated by PCBs in PC12 cells. Of the congeners tested, ortho-substituted PCBs were found to induce PLA2 activation. PLA2 activation by 2,2′,4,6-tetrachlorobiphenyl (TeCB), the most potent congener, was inhibited by bromoenol lactone (BEL), a calcium-independent PLA2 (iPLA2) inhibitor, and methyl arachidonyl fluorophosphonate (MAFP), a cytosolic PLA2 and iPLA2 inhibitor. In the case of Ca2+, although 2,2′,4,6-TeCB increased [Ca2+]i in the presence of extracellular Ca2+, PLA2 activation was not inhibited by EGTA and 1,2-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxy-methyl) ester, an extracellular and an intracellular Ca2+ chelator, respectively. On the other hand, 2,2′,4,6-TeCB-induced Ca2+ increase was partially inhibited by BEL and MAFP. In addition, 2,2′,4,6-TeCB induced apoptotic cell death in these cells. Taken together, our results suggest that ortho-substituted PCBs might induce apoptosis through PLA2-mediated Ca2+ influx, which provides a clue to understand the mechanism of neurotoxic effects of ortho-substituted PCBs.

Effect of lactational exposure to 1,2,3,4- tetrachlorodibenzo-p-dioxin on cytochrome P-450 1A1 mRNA in the neonatal rat liver: quantitative analysis by the competitive RT-PCR method.

The aim of this study was to assess the effect of lactational exposure to dioxins in neonates on the cytochrome P450 1A1 (CYP1A1) induction in the level of gene expression. Maternal rats were treated with a single dose of 50 or 100 micromol/kg 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD), a low potent congener of dioxins, on the first day post-partum (day 1). Induction of CYP1A1 mRNA expression was quantitatively analyzed by the competitive reverse transcription-polymerase chain reaction (RT-PCR) method. The CYP1A1 mRNA was detectable at extremely low amounts in the liver of control neonates and mothers. The mRNA ratios of CYP1A1 to beta-actin in neonates were dose-dependently increased by the treatment of 1,2,3,4-TCDD of their mothers. Its peak occurred on day 6 and was sustained at the same level on day 10. Increases of the ratio with 100 micromol/kg 1,2,3,4-TCDD on day 2, 6 and 10 were 26-, 40- and 40-fold of the appropriate controls, respectively. These levels paralleled the activity of ethoxyresorufin-o-deethylase, representing CYP1A mediated monooxygenase. In the mother, the mRNA ratio was increased only to threefold of the control, 10 days after treatment. Current RT-PCR procedure enabled to assess both constitutive and induced levels of CYP1A1 mRNA in the neonatal rat livers. Although the dose of 1,2,3,4-TCDD selected in this study was about 5000 times higher than the daily intake of dioxins in breast-fed infants, CYP1A1 mRNA was highly induced for a longer period of time in neonatal rats receiving 1,2,3,4-TCDD via lactation than the treated maternal rats.

Relative potencies of individual polychlorinated naphthalenes to induce dioxin-like responses in fish and mammalian in vitro bioassays.

A growing body of evidence suggests that polychlorinated naphthalenes (PCNs) may be fairly widespread environmental contaminants. This may be cause for concern because exposure to PCNs has been linked to dioxin-like biological responses in a wide variety of species. This study used three in vitro bioassays to characterize the dioxin-like potency of 18 individual PCN congeners and 1 PCN metabolite. The PLHC-1 fish hepatoma cell bioassay was relatively insensitive to PCNs. At the concentrations tested, only 1, 4 di-CN and 2,4-dichloro-1-napthol caused significant induction of ethoxyresorufin O-deethylase (EROD) activity in the PLHC-1 assay. In vitro EROD and luciferase assays using recombinant H4IIE rat hepatoma cells were more responsive to PCNs. Structure-activity relationships were observed both in terms of the degree of chlorination and the positions of chlorine substitutions. Hexa-chlorinated naphthalenes (CNs), exhibiting relative potencies (REPs) around 10(-3) (relative to TCDD), were the most potent congeners tested. Penta-CNs were also rather potent, yielding REPs between 10(-3) and 10(-7). Tetra-, tri-, di-, and mono-CNs were less active. REPs for the active congeners were similar to those for some PCBs. The relative potency estimates reported here contribute to an emerging body of information that will aid determination of the relative contribution of PCNs to the total dioxin-like activity associated with environmental samples.

Structure dependent induction of CYP1A by polychlorinated biphenyls in hepatocytes of male castrated pigs

Hepatocytes cultures prepared from castrated pig hepatocytes (Great Yorkshire × Dutch Landrace), as a model for human liver, were used to study the effect of twenty polychlorinated biphenyls (PCBs) on CYP1A activity, measured as the dealkylation of either ethoxyresorufin or methoxyresorufin. The selection of the PCBs was based on their differences in physico-chemical properties. The non- ortho and mono- ortho substituted PCBs were the most potent CYP1A inducers in pig hepatocytes. In addition, several multiple- ortho substituted congeners, with five or more chlorine atoms, were inducers of CYP1A activity as well. Their relative effect potencies (REP) were proximately 10,000 times lower than the most potent congener, 3,3′,4,4′,5 PeCB (PCB#126). Using partial least-squares (PLS) modeling, predictions of CYP1A activity could be made for all tetra to hepta substituted congeners. Several multiple- ortho substituted PCBs, which are highly abundant in the biotic and abiotic environment, have been found to induce CYP1A activity in pig hepatocytes. Because induction of CYP1A activity is used as biomarker for Ah-receptor mediated responses, it is suggested to include these congeners in future risk assessment.

Relative Potencies of Individual Polychlorinated Naphthalenes and Halowax Mixtures To Induce Ah Receptor-Mediated Responses

Polychlorinated naphthalenes (PCNs) are ubiquitous environmental pollutants that are structurally similar to other polychlorinated diaromatic hydrocarbons (PCDHs), such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs). Despite being ubiquitous, much less is known about the fate, transport, and biological effects of individual PCN congeners than other PCDHs. The purpose of the current study was to utilize an in vitro assay (H4IIE-luc) to determine potencies relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 20 individual PCNs (from 75 possible congeners) and 6 Halowax mixtures. H4IIE rat hepatoma cells (H4IIE-Luc), which are stably transfected with an Ah receptor (AhR)-controlled luciferase reporter gene construct, respond specifically to AhR agonists and are thus a reasonable measure of AhR-mediated, or dioxin-like, activity. The most potent congeners were 1,2,3,4,6,7-hexa-CN (PCN 66), 1,2,3,5,6,7-hexa-CN (PCN 67), and 1,2,3,4,5,6,7-hepta-CN (PCN 73), with ...

Multivariate modeling of polychlorinated biphenyl–induced CYP1A activity in hepatocytes from three different species: Ranking scales and species differences

AbstractCytochrome P4501A–induced activity of 20 selected polychlorinated biphenyls (PCBs) was evaluated by measuring ethoxyresorufin‐O‐deethylase and methoxyresorufin‐O‐demethylase activities induced in the hepatocytes of cynomolgus monkeys, male castrated pigs, and chicken embryos. Quantitative structure‐activity relationships have been established, including 52 physi‐cochemical parameters and different measures of the dose‐response curves. Relative effect potencies are predicted for the 154 tetra‐to hepta‐PCBs and reported for the most potent congeners according to both EC50 and maximal response values. Important physicochemical parameters of the PCBs as related to the modeled activity are parts of their ultraviolet absorption spectra, the Henry's law constant, the ionization potential, and the octanol‐water partition coefficient. Interspecies differences were found in terms of varied sensitivity to different structural subgroups of the compounds. The chicken hepatocyte assay showed the most specific structure‐activity relationship, with high activity for the non‐ortho PCBs, whereas the pig hepatocytes responded even for some di‐ to tetra‐ortho PCBs. An interspecies response, the principal induction potency, is presented for the 41 most potent PCBs. These responses showed strong correlation with the toxic equivalency factors and are likely to be useful in risk assessment of the compounds.

Structure-Dependent Induction of CYP1A by Polychlorinated Biphenyls in Hepatocytes of Cynomolgus Monkeys (Macaca fascicularis)

Until now structure–activity relationships (SARs) forin vitroorin vivoCYP1A induction by polychlorinated biphenyls (PCBs) have only been determined in rodents and birds. This study describes the first development of such a SAR in a primate species by using hepatocyte cultures of cynomolgus monkey (Macaca fascicularis). Hepatocyte cultures of primate species might be a more suitable model for humans than those of rodents. For 20 PCBs, thein vitroinduction of CYP1A activity was determined by measuring dealkylation of either methoxyresorufin or ethoxyresorufin. Selection of PCBs was based on multivariate physical–chemical characterization of all tetra- through heptachlorinated congeners. The non-ortho-substituted congeners were found to be the most potent inducers, followed by the mono-ortho-substituted PCBs. Multiple-ortho-substituted congeners, with more than five chlorine atoms, were inducers of CYP1A activity in monkey hepatocytes as well, with EC50 values approximately 10,000 times higher than 3,3′,4,4′,5 PeCB (PCB 126), the most potent congener. Using partial least-squares (PLS) modeling, predictions of CYP1A activity were established for all other tetra- to hepta-substituted congeners. Several congeners, which are abundant in the (a)biotic environment, were predicted to have CYP1A activity in cynomolgus monkey hepatocytes. Because induction of CYP1A activity is generally used as an early and sensitive biomarker for theAh-receptor-mediated potential of a chemical, further studies are recommended to determine the possible risks of these multiple-orthoPCBs to humans.

Multiple Limbic Regions Mediate the Disruption of Prepulse Inhibition Produced in Rats by the Noncompetitive NMDA Antagonist Dizocilpine

Prepulse inhibition (PPI), a phenomenon in which a weak prestimulus decreases the startle response to an intense stimulus, provides an operational measure of sensorimotor gating (a process by which an organism filters sensory information) and is diminished in schizophrenia and schizotypal patients. The psychotomimetic phencyclidine and its potent congener dizocilpine are noncompetitive antagonists of the NMDA receptor complex, and they disrupt PPI in rodents, mimicking the clinically observed PPI deficit. The neuroanatomical substrates mediating the PPI-disruptive effects of noncompetitive NMDA antagonists are unknown. The present study sought to identify brain regions subserving the disruption of PPI produced by noncompetitive NMDA antagonists in rats. PPI was measured in startle chambers immediately after bilateral infusion of dizocilpine (0, 0.25, 1.25, and 6.25 microgram/0.5 microliter/side) into one of six brain regions: amygdala, dorsal hippocampus, medial prefrontal cortex, nucleus accumbens, ventral hippocampus, and dorsomedial thalamus. Dizocilpine significantly decreased PPI after infusion into the amygdala or dorsal hippocampus. A trend toward PPI disruption was observed with administration into medial prefrontal cortex. In contrast, no change in PPI was produced by dizocilpine infusion into nucleus accumbens, ventral hippocampus, or dorsomedial thalamus. Startle reactivity was increased by dizocilpine infusion into amygdala, dorsal hippocampus, nucleus accumbens, and dorsomedial thalamus, but not medial prefrontal cortex. These findings indicate that multiple limbic forebrain regions mediate the ability of noncompetitive NMDA antagonists to disrupt PPI and that the PPI-disruptive and the startle-increasing effects of dizocilpine are mediated by different central sites.

Broad-spectrum antiviral activity and mechanism of antiviral action of the fluoroquinolone derivative K-12.

The fluoroquinolone derivatives have been shown to inhibit human immunodeficiency virus (HIV) replication at the transcriptional level. We confirmed the anti-HIV activity of the most potent congener, 8-difluoromethoxy-1-ethyl-6-fluoro-1,4-dihydro-7-[4-(2- methoxyphenyl)-1-piperazinyl]-4-quinolone-3-carboxylic acid (K-12), in both acutely and chronically infected cells. K-12 was active against different strains of HIV-1 (including AZT- and ritonavir-resistant HIV-1 strains), HIV-2 and simian immunodeficiency virus, in MT-4, CEM, C8166 and peripheral blood mononuclear cells. In all of these antiviral assay systems, K-12 showed a similar activity (EC50 0.2-0.6 microM). K-12 inhibited Moloney murine sarcoma virus-induced transformation of C3H/3T3 cells with an EC50 of 6.9 microM. Also, K-12 proved inhibitory to herpesvirus saimiri, human cytomegalovirus, varicella-zoster virus and herpes simplex virus types 1 and 2 (in order of decreasing sensitivity), but was not inhibitory (at subtoxic concentrations) to human herpesvirus type 8 (as evaluated in BCBL-1 cells), vaccinia virus, Sindbis virus, vesicular stomatitis virus, respiratory syncytial virus, Coxsackie virus, Punta Toro virus, parainfluenza virus or reovirus. Time-of-addition experiments and quantitative transactivation bioassays indicated that K-12 inhibits the Tat-mediated transactivation process in HIV-infected cells.

Transcriptional suppression of estrogen receptor gene expression by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)

TCDD, the most potent congener of the polychlorinated dioxins, has been shown to be an antiestrogen. The mechanisms of TCDD-induced antiestrogenicity are still under investigation. In this study, we investigated the effects of TCDD on the expression of the estrogen receptor (ER) gene. We studied the levels of un-spliced ER transcript (hnRNA) as well as the ER mRNA in ovary, uterus and liver of TCDD-treated mice with different genetic backgrounds. To quantitate the ER hnRNA levels, the intron and exon boundary of ER hnRNA was amplified by competitive RT-PCR. The ER mRNA from these mice was quantitated by competitive RT-PCR amplifying exons separated by an intron. ER hnRNA and ER mRNA levels were quantitated 4 days after a single i.p. dose of TCDD (5 μg/kg) in female C57BL/6J (B6) mice, which carry the responsive allele to TCDD. TCDD treatment significantly ( p<0.05) suppressed the levels of ER hnRNA in the ovary (27.4%) and uterus (21.9%). The decreases in ER hnRNA were coordinated with significant ( p<0.01) decreases in ER mRNA in ovary (57.7%) and uterus (37.6%). There was a significant decrease (20.3%, p<0.05) in liver ER mRNA, however, the changes of ER hnRNA in liver were not significant. The coordinated decreases in ER hnRNA and mRNA in TCDD-treated mice suggest a suppression of transcription of the ER gene. We performed the same study on DBA/2J (D2) mice, which possess the “non-responsive” allele of the aryl hydrocarbon receptor (AhR). These mice demonstrated no significant decrease in either the ER mRNA or hnRNA after TCDD treatment. Overall, these results suggest that TCDD suppresses the gene expression of the ER receptor by decreasing its transcription, and the AhR plays an important role in mediating this response.