Analysis for data-derived extrapolation factors for procymidone.

The derivation of Chemical Specific Adjustment Factors (CSAFs) (IPCS, 2005; U.S. EPA, 2014) depends on the choice of appropriate dose metric. EPA and IPCS guidance was applied to derive a CSAF for developmental toxicity for procymidone (PCM). Although kinetic data were not available in humans at any dose, sufficient toxicokinetic data are available in a surrogate species, primates, and from chimeric mice with both rat and human liver cells to offer insights. Alternative approaches were explored in the derivation of the CSAG based on review of the available kinetic data. The most likely dosimetric adjustment is the Cmax based on the character of the critical effect - reduced anogenital distance and increased incidence of hypospadias in male rats, which likely occurs during a small window of time during development of the rat fetus. Cmax is also the default dosimeter from U.S. EPA (1991). However, in this case, the use of Cmax is also likely more conservative than the use of area under the curve (AUC), which otherwise is the default recommendation of the IPCS (2005). Despite human data, estimated tentative CSAF value is 0.48 (range, 0.22 to 0.74). The use of any of these values would be supported by the available data.

Developmental toxicity of procymidone to larval zebrafish based on physiological and transcriptomic analysis.

As a broad-spectrum with low toxicity, procymidone (PCM), is widely used in agriculture and frequently observed in aquatic system, which may cause some impacts on aquatic organisms. Here, to determine the developmental toxicity of PCM, embryonic and larval zebrafish were exposed to PCM at 0, 1, 10, 100 µg/L in dehydrogenated natural water containing 0.01% acetone for 7 days. The results showed that high concentration of PCM could cause the pericardial edema and increase the heart rates in larval zebrafish, suggesting that PCM had developmental toxicity to zebrafish. We also observed that PCM exposure not only changed the physiological parameters including TBA, GLU and pyruvic acid, but also changed the transcriptional levels of glycolipid metabolism related genes. In addition, after transcriptomics analysis, a total of 1065 differentially expressed genes, including 456 up-regulated genes and 609 down-regulated genes, changed significantly in 100 µg/L PCM treated larval zebrafish. Interestingly, after GO (Gene Ontology) analysis, the different expression genes (DEGs) were mainly enriched to the three different biology processes including GABA-nervous, lipid Metabolism and response to drug. We also observed that the levels of GABA receptor related genes including gabrg2, gabbr1α, gabbr1 and gabra6α were inhibited by PCM exposure. Interestingly, the swimming distance of larval zebrafish had the tendency to decrease after PCM exposure, indicating that the nervous system was affected by PCM. Taken together, the results confirmed that the fungicide PCM could cause developmental toxicity by influencing the lipid metabolism and GABA mediated nervous system and behavior in larval zebrafish. We believed that the results could provide an important data for the influence of PCM on aquatic animals.

The Inactivation by Curcumin-Mediated Photosensitization of Botrytis cinerea Spores Isolated from Strawberry Fruits.

Photosensitization is a novel environmentally friendly technology with promising applications in the food industry to extend food shelf life. In this study, the natural food dye curcumin, when combined with visible light (430 nm), was shown to be an effective photosensitizer against the common phytopathogenic fungi Botrytis cinerea (the cause of grey mould). Production of the associated phytotoxic metabolites botrydial and dihydrobotrydial was measured by our newly developed and validated HRAM UPLC-MS/MS method, and was also shown to be reduced by this treatment. With a light dose of 120 J/cm2, the reduction in spore viability was directly proportional to curcumin concentrations, and the overall concentration of both botrydial and dihydrobotrydial also decreased with increasing curcumin concentration above 200 µM. With curcumin concentrations above 600 µM, the percentage reduction in fungal spores was close to 100%. When the dye concentration was increased to 800 µM, the spores were completely inactive and neither botrydial nor dihydrobotrydial could be detected. These results suggest that curcumin-mediated photosensitization is a potentially effective method to control B. cinerea spoilage, and also to reduce the formation of these phytotoxic botryane secondary metabolites.

Quantitative in Vitro to in Vivo Extrapolation (QIVIVE) for Predicting Reduced Anogenital Distance Produced by Anti-Androgenic Pesticides in a Rodent Model for Male Reproductive Disorders.

BACKGROUND: Many pesticides can antagonize the androgen receptor (AR) or inhibit androgen synthesis in vitro but their potential to cause reproductive toxicity related to disruption of androgen action during fetal life is difficult to predict. Currently no approaches for using in vitro data to anticipate such in vivo effects exist. Prioritization schemes that limit unnecessary in vivo testing are urgently needed. OBJECTIVES: The aim was to develop a quantitative in vitro to in vivo extrapolation (QIVIVE) approach for predicting in vivo anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats. METHODS: We built a physiologically based pharmacokinetic (PBK) model to simulate concentrations of chemicals in the fetus resulting from maternal dosing. The predicted fetal levels were compared with analytically determined concentrations, and these were judged against in vitro active concentrations for AR antagonism and androgen synthesis suppression. RESULTS: We first evaluated our model by using in vitro and in vivo anti-androgenic data for procymidone, vinclozolin, and linuron. Our PBK model described the measured fetal concentrations of parent compounds and metabolites quite accurately (within a factor of five). We applied the model to nine current-use pesticides, all with in vitro evidence for anti-androgenicity but missing in vivo data. Seven pesticides (fludioxonil, cyprodinil, dimethomorph, imazalil, quinoxyfen, fenhexamid, o-phenylphenol) were predicted to produce a shortened AGD in male pups, whereas two (λ-cyhalothrin, pyrimethanil) were anticipated to be inactive. We tested these expectations for fludioxonil, cyprodinil, and dimethomorph and observed shortened AGD in male pups after gestational exposure. The measured fetal concentrations agreed well with PBK-modeled predictions. DISCUSSION: Our QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as in vivo anti-androgens. With the examples investigated, our approach shows great promise for predicting in vivo anti-androgenicity (i.e., AGD shortening) for chemicals with in vitro activity and for minimizing unnecessary in vivo testing. https://doi.org/10.1289/EHP6774.

Structures of N-Hydroxy-Type Tetrodotoxin Analogues and Bicyclic Guanidinium Compounds Found in Toxic Newts.

The biosynthesis of tetrodotoxin (TTX, 1), a potent neurotoxin widely distributed in marine and terrestrial metazoans, remains unresolved. A significant issue has been identifying intermediates and shunt products associated with the biosynthetic pathway of TTX. We investigated TTX biosynthesis by screening and identifying new TTX-related compounds from Cynops ensicauda popei and Taricha granulosa. Mass spectrometry (MS)-guided screening identified two new N-hydroxy TTX analogues in newts: 1-hydroxy-8-epiTTX (2) and 1-hydroxy-8-epi-5,11-dideoxyTTX (3, previously reported as 1-hydroxy-5,11-dideoxyTTX). We prepared a new analogue, 8-epi-5,11-dideoxyTTX (4), from 3 via N-OH reduction and confirmed the presence of 4 in T. granulosa using hydrophilic interaction liquid chromatography (HILIC)-LCMS. The presence of 8-epi-type TTX analogues in both Cynops and Taricha supports a branched biosynthetic pathway of terrestrial TTX, which produces 6- and 8-epimers. In addition, new bicyclic guanidinium compounds Tgr-238 (5) and Tgr-240 (6) were identified as putative shunt products of our proposed TTX biosynthesis pathway. A structural analysis of Cep-228A (7), another bicyclic compound, was performed using NMR. Based on the structures of 5-7 and their analogues, we propose a model of the shunt and metabolic pathways of the terrestrial TTX biosynthesis.

Genotoxic effect of 2,2'-bis(bicyclo[2.2.1] heptane) on bacterial cells.

The toxic effect of strained hydrocarbon 2,2'-bis (bicyclo[2.2.1]heptane) (BBH) was studied using whole-cell bacterial lux-biosensors based on Escherichia coli cells in which luciferase genes are transcriptionally fused with stress-inducible promoters. It was shown that BBH has the genotoxic effect causing bacterial SOS response however no alkylating effect has been revealed. In addition to DNA damage, there is an oxidative effect causing the response of OxyR/S and SoxR/S regulons. The most sensitive to BBH lux-biosensor was E. coli pSoxS-lux which reacts to the appearance of superoxide anion radicals in the cell. It is assumed that the oxidation of BBH leads to the generation of reactive oxygen species, which provide the main contribution to the genotoxicity of this substance.

Nonclassical Phenyl Bioisosteres as Effective Replacements in a Series of Novel Open-Source Antimalarials.

The replacement of one chemical motif with another that is broadly similar is a common method in medicinal chemistry to modulate the physical and biological properties of a molecule (i.e., bioisosterism). In recent years, bioisosteres such as cubane and bicyclo[1.1.1]pentane (BCP) have been used as highly effective phenyl mimics. Herein, we show the successful incorporation of a range of phenyl bioisosteres during the open-source optimization of an antimalarial series. Cubane (19) and closo-carborane (23) analogues exhibited improved in vitro potency against Plasmodium falciparum compared to the parent phenyl compound; however, these changes resulted in a reduction in metabolic stability; unusually, enzyme-mediated oxidation was found to take place on the cubane core. A BCP analogue (22) was found to be equipotent to its parent phenyl compound and showed significantly improved metabolic properties. While these results demonstrate the utility of these atypical bioisosteres when used in a medicinal chemistry program, the search to find a suitable bioisostere may well require the preparation of many candidates, in our case, 32 compounds.

Diterpenes with bicyclo[2.2.2]octane moieties from the fungicolous fungus Xylaria longipes HFG1018.

Xylarilongipins A (1) and B (2), two diterpenes each with an unusual cage-like bicyclo[2.2.2]octane moiety, along with their biosynthetic precursor hymatoxin L (3), were isolated from the culture broth of the fungicolous fungus Xylaria longipes HFG1018 inhabiting in the medicinal fungus Fomitopsis betulinus. The structures and absolute configurations of the three compounds were established by extensive spectroscopic analysis and single-crystal X-ray diffraction analysis. Xylarilongipin A (1) displayed moderate inhibitory activity against the cell proliferation of concanavalin A-induced T lymphocytes and lipopolysaccharide-induced B lymphocytes with IC50 values of 13.6 and 22.4 µM, respectively. Additionally, the biosynthetic pathways for compounds 1-3 are discussed. This work not only corroborates the structure of the 9,16-cyclo-(18-nor-)isopimarane skeleton by single-crystal X-ray diffraction analysis for the first time, but also provides new insights into the biosynthetic origin of the unusual diterpene skeletons.

Toxicological effects of comercial formulations of fungicides based on procymidone and iprodione in seedlings and root tip cells of Allium cepa.

In this study the phytotoxic, cytotoxic, genotoxic and mutagenic effects of two commercial fungicide-active compounds, procymidone (PR) and iprodione (IP), were determined. The parameters evaluated were germination and root growth, mitotic index, chromosomal and nuclear aberrations, and molecular analyses were also performed in the model plant Allium cepa L. The results demonstrated that the active compounds PR and IP were phytotoxic, delaying germination and slowing the development of A. cepa seedlings. Moreover, PR and IP showed cytogenotoxicity towards A. cepa meristematic cells, inducing chromosomal changes and cell death. The mutagenic activity of the active compounds was demonstrated by the detection of DNA changes in simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers in the treated cells compared to the negative control. Together, these results contribute to a better understanding of the damage caused by these substances in living organisms and reveal a promising strategy for prospective studies of the toxic effects of environmental pollutants.

Comparative study of alpha- and beta-pinene effect on PTZ-induced convulsions in mice.

Convulsions occur in response to a loss of balance between excitatory and inhibitory neurotransmitters, and the treatment for this condition consists in restore such lost balance. Many anticonvulsant drugs present side effects which may limit their use. This fact has stimulated the search for new sources of treatment from aromatic plants. Many monoterpenes commonly present in essential oils are known because of their anticonvulsant properties. The anticonvulsant effect of α- and ß-pinene, two structural isomers, is still little studied. Thus, the present work evaluated the anticonvulsant effect of α- and ß-pinene in pentylenetetrazole-induced convulsions model. Initially, the oral LD50 for α- and ß-pinene was estimated. Following the oral administration, a mild sedation was observed and no deaths were recorded; the LD50 estimated for both monoterpenes was greater than 2 000 mg/kg, p.o. Further, animals were orally treated with α-pinene (100, 200 and 400 mg/kg), ß-pinene (100, 200 and 400 mg/kg) and the equimolar mixture of α- and ß-pinene (400 mg/kg) and subjected to the pentylenetetrazole-induced convulsions model. In this model, only the dose of 400 mg/kg of the compounds was able to significantly decrease the seizure intensity. The latency of first convulsion was significantly increased by the mixture of α- and ß-pinene (400 mg/kg). In addition, ß-pinene and the mixture of the two monoterpenes, both at a dose of 400 mg/kg, significantly increased the time of death of animals. The treatment with ß-pinene and the equimolar mixture of the two monoterpenes significantly reduced hippocampal nitrite level and striatal content of dopamine (DA) and norepinephrine (NE). Taken together, the results suggest that α-pinene appears to be devoid of anticonvulsant action. This fact, however, seems to be dependent on the chemical structure of the compound, since pretreatment with the ß-pinene increased the time of death pf PTZ-treated mice, which seems to depend on the ability of the compound to reduce nitrite concentration and NE and DA content, during the pentylenetetrazole-induced seizure.

Identification of Metabolism and Excretion Differences of Procymidone between Rats and Humans Using Chimeric Mice: Implications for Differential Developmental Toxicity.

A metabolite of procymidone, hydroxylated-PCM, causes rat-specific developmental toxicity due to higher exposure to it in rats than in rabbits or monkeys. When procymidone was administered to chimeric mice with rat or human hepatocytes, the plasma level of hydroxylated-PCM was higher than that of procymidone in rat chimeric mice, and the metabolic profile of procymidone in intact rats was well reproduced in rat chimeric mice. In human chimeric mice, the plasma level of hydroxylated-PCM was less, resulting in a much lower exposure. The main excretion route of hydroxylated-PCM-glucuronide was bile (the point that hydroxylated-PCM enters the enterohepatic circulation) in rat chimeric mice, and urine in human chimeric mice. These data suggest that humans, in contrast to rats, extensively form the glucuronide and excrete it in urine, as do rabbits and monkeys. Overall, procymidone's potential for causing teratogenicity in humans must be low compared to that in rats.