Integrated testing and assessment approaches for skin sensitization: a commentary.

A Bayesian integrated testing strategy (ITS) approach, aiming to assess skin sensitization potency, has been presented, in which data from various types of in vitro assays are integrated and assessed in combination for their ability to predict in vivo skin sensitization data. Here we discuss this approach and compare it to our quantitative mechanistic modeling (QMM) approach based on physical organic chemistry. The main findings of the Bayesian study are consistent with our chemistry-based approach and our previously published assessment of the key determinants of sensitization potency, in particular the relatively high predictive value found for chemical reactivity data and the relatively low predictive value for bioavailability parameters. As it stands at present the Bayesian approach does not utilize the full range of predictive capability that is already available, and aims only to assign potency categories rather than numerical potency values per se. In contrast, for many chemicals the QMM approach can already provide numerical potency predictions. However, the Bayesian approach may have potential for those chemicals where a chemistry modeling approach cannot provide a complete answer (e.g. pro-electrophiles whose in cutaneo activation cannot currently be modeled confidently). Nonetheless, our main message is of the importance of leveraging chemistry insights and read-across approaches to the fullest extent possible.

Relative susceptibility and transcriptional response of nitrogen cycling bacteria to quantum dots.

Little is known about the potential impacts of accidental or incidental releases of manufactured nanomaterials to microbial ecosystem services (e.g., nutrient cycling). Here, quantum dots (QDs) coated with cationic polyethylenimine (PEI) were more toxic to pure cultures of nitrogen-cycling bacteria than QDs coated with anionic polymaleic anhydride-alt-1-octadecene (PMAO). Nitrifying bacteria (i.e., Nitrosomonas europaea) were much more susceptible than nitrogen fixing (i.e., Azotobacter vinelandii, Rhizobium etli, and Azospirillum lipoferum) and denitrifying bacteria (i.e., Pseudomonas stutzeri). Antibacterial activity was mainly exerted by the QDs rather than by their organic coating or their released QD components (e.g., Cd and Zn), which under the near-neutral pH tested (to minimize QD weathering) were released into the bacterial growth media at lower levels than their minimum inhibitory concentrations. Sublethal exposure to QDs stimulated the expression of genes associated with nitrogen cycling. QD-PEI (10 nM) induced three types of nitrogenase genes (nif, anf, and vnf) in A. vinelandii, and one ammonia monooxygenase gene (amoA) in N. europaea was up-regulated upon exposure to 1 nM QD-PEI. We previously reported up-regulation of denitrification genes in P. stutzeri exposed to low concentrations of QD-PEI. (1) Whether this surprising stimulation of nitrogen cycling activities reflects the need to generate more energy to overcome toxicity (in the case of nitrification or denitrification) or to synthesize organic nitrogen to repair or replace damaged proteins (in the case of nitrogen fixation) remains to be determined.

Facilitated internalization of neocarzinostatin and its lipophilic polymer conjugate, SMANCS, into cytosol in acidic pH.

The effects of environmental pH on the binding and cytotoxicity of the antitumor proteins neocarzinostatin (NCS) and SMANCS [copoly(styrene-maleic acid)-conjugated NCS] to cultured cells were studied by using their fluorescent-labeled derivatives (F-drugs). At 37 degrees C the binding of these drugs to HeLa cells was pH dependent: The amount of cell-bound drugs increased with an increase in the acidity of the medium. The pH-dependent change in the binding of the drugs was not as evident at 0 degree C. The cytotoxic action of these drugs was much more rapid at acidic pH compared with that at neutral or slightly alkaline pH. Furthermore, F-drugs could be utilized to probe the microenvironmental pH in Meth-A cells, in which the drug was located by the ratio of fluorescent intensities at 450 and 490 nm. The environment of the cell-bound F-drugs became acidic with incubation time at 37 degrees C but not at 0 degree C. Inasmuch as these drugs directly attack DNA, these results suggest that NCS and SMANCS are translocated across the membrane of acidic vesicles into the cytosol after endocytotic uptake. This hypothesis is also supported by the finding that NH4Cl and chloroquine protected HeLa cells against the cytotoxicity of the drugs. Data also showed that the hydrophobic polyanion conjugate SMANCS had a much greater cell binding (10 times) and more rapid internalization compared with NCS. Taken together, our results show that acidic pH of tumor tissue is preferable for effective binding and internalization into cytosol for NCS and SMANCS.

Antitumor effects of SMANCS on rat mammary tumor induced by 7,12-dimethylbenz[a]anthracene.

We previously found that a high-molecular-weight anticancer agent, polystyrene-co-maleic acid conjugated neocarzinostatin (SMANCS), in which two chains of styrene/maleic acid copolymer are conjugated to the anticancer protein neocarzinostatin (NCS), accumulated more selectively in tumor tissue than in normal tissue and was more stable than NCS in blood. These results indicate that SMANCS should have less systemic toxicity and a better therapeutic effect than NCS. In this study, the antitumor activity and adverse effects of SMANCS were compared with those of NCS by using rat mammary tumor induced by 7,12-dimethylbenz[a]anthracene. When tumors of rats, that had received 7,12-dimethylbenz[a]anthracene (20 mg/kg, one dose, p.o. in oily formulation), became palpable usually after 4-20 weeks, SMANCS treatment was initiated. Thirty days after i.v. administration of SMANCS (0.1 mg/kg 3 times and 0.3 mg/kg 3 times), tumors had shrunk in 35 of 37 rats (a mean weight was about 10% of control value; or decreased to about 30% of the value of before treatment in tumor weight); tumor size had not changed in 1 rat, and in the remaining 1 rat the tumor had enlarged. Thirty days after i.v. administration of NCS, tumors had shrunk in 8 of 14 rats, but the tumor size was unchanged in 1 rat and was enlarged in 5. In the control group, all tumors had enlarged. Development of new tumors was completely prevented by the administration of SMANCS. Histological examination of sequential slices of tumor revealed clear finding of degeneration and tumor encapsulation at 30 days after initial administration of SMANCS, with an accompanying fatty degeneration, but these effects were not observed for tumors treated with NCS. Although red blood cell counts and hemoglobin amounts decreased significantly in rats receiving NCS, no such effects were apparent in the SMANCS group.

High-Content Imaging and Gene Expression Approaches To Unravel the Effect of Surface Functionality on Cellular Interactions of Silver Nanoparticles.

The toxic effects of Ag nanoparticles (NPs) remain an issue of debate, where the respective contribution of the NPs themselves and of free Ag(+) ions present in the NP stock suspensions and after intracellular NP corrosion are not fully understood. Here, we employ a recently set up methodology based on high-content (HC) imaging combined with high-content gene expression studies to examine the interaction of three types of Ag NPs with identical core sizes, but coated with either mercaptoundecanoic acid (MUA), dodecylamine-modified poly(isobutylene-alt-maleic anhydride) (PMA), or poly(ethylene glycol) (PEG)-conjugated PMA with two types of cultured cells (primary human umbilical vein endothelial cells (HUVEC) and murine C17.2 neural progenitor cells). As a control, cells were also exposed to free Ag(+) ions at the same concentration as present in the respective Ag NP stock suspensions. The data reveal clear effects of the NP surface properties on cellular interactions. PEGylation of the NPs significantly reduces their cellular uptake efficiency, whereas MUA-NPs are more prone to agglomeration in complex tissue culture media. PEG-NPs display the lowest levels of toxicity, which is in line with their reduced cell uptake. MUA-NPs display the highest levels of toxicity, caused by autophagy, cell membrane damage, mitochondrial damage, and cytoskeletal deformations. At similar intracellular NP levels, PEG-NPs induce the highest levels of reactive oxygen species (ROS), but do not affect the cell cytoskeleton, in contrast to MUA- and PMA-NPs. Gene expression studies support the findings above, defining autophagy and cell membrane damage-related necrosis as main toxicity pathways. Additionally, immunotoxicity, DNA damage responses, and hypoxia-like toxicity were observed for PMA- and especially MUA-NPs. Together, these data reveal that Ag(+) ions do contribute to Ag NP-associated toxicity, particularly upon intracellular degradation. The different surface properties of the NPs however result in distinct toxicity profiles for the three NPs, indicating clear NP-associated effects.

Cytotoxicity of smancs in comparison with other anticancer agents against various cells in culture.

The cytoxicity of neocarzinostatin (NCS) and smancs [copoly(styrene maleic acid)-conjugated NCS] to various cultured cells was compared with that of several other antitumor agens in clinical use on various malignant and non-malignant cells as regards to their effect on colony formation of cells. Both NCS and smancs showed the most potent cytotoxicity against all tumor cell lines tested; the IC50s (colony inhibitory concentration 50%) of these drugs were 3.2-20 nM, 10-1000 times lower than those of other drugs. In contrast, NCS and smancs exhibited relatively lower toxicity to normal cells such as human skin fibroblasts and chick embryonic fibroblasts (IC50, about 50 and 100 nM, respectively). Normal rat hepatocytes were found to be very resistant to NCS and smancs (both IC50s were about 500 nM). Moreover, the minimum exposure time of smancs to cultured tumor cells required to achieve effective cytotoxic activity was much shorter than that of NCS and other drugs. Namely, at 30 nM more than 80% cells were killed by exposure to smancs for only a few minutes, whereas with NCS more than 80 min of exposure time was required. It was also found that smancs inhibited the uptake of 3H-thymidine into DNA as expected. These results clearly indicate that smancs is an unique antitumor agent with a broad antitumor spectrum which exhibits some characteristics similar to, but also some very different from NCS.

A quantitative comparison of induction and challenge concentrations inducing a 50% positive response in three skin sensitization tests; the guinea pig maximization test, adjuvant and patch test and Buehler test.

The sensitivities of three skin sensitization tests such as the Guinea pig maximization test (GPMT), Adjuvant and patch test (APT) and Buehler test (BT), were quantitatively compared with reference to induction and challenge concentrations. Four chemical which had different physico-chemical properties (octanol-water partition coefficient (logP) and reactivity with NH2-group) were used in order to clarify the effect of the physico-chemical properties of chemicals on the sensitivity of the different methods. The induction concentrations inducing a 50% positive response (IC50) demonstrated extreme variation with the three methods. For example, the BT/GPMT ratio of IC50 values for 2,4-dinitrochlorobenzene was 33, whereas that for maleic anhydride was 300,000. The results were thought to be caused by difference properties such as the logP and reactivity of chemicals. This correlation was confirmed by using 2-dodecen-1-yl succinic anhydride, which had the same reactivity but higher logP than that of maleic anhydride. On the other hand, the challenge concentrations inducing 50% positive responses (CC50) were less affected by the methods and the BT/GPMT ratios for CC50 values were all within a 10-fold range. These results suggest that the sensitivity might be strongly different with reference to induction concentration, but not challenge concentration among the three methods.

Long-term toxicity studies of styrene maleic anhydride in rats.

In male Charles Foster rats, polymer styrene maleic anhydride was injected into the lumen of the vas deferens at dose levels of 1.0, 2.5, and 5.0 mg in each vas deferens of Groups II, III, and IV, respectively, while controls (Group I) received 0.03 ml dimethyl sulfoxide in each vas deferens. The rats were observed for 6 months for toxicity. No change in any of the toxicity parameters in test animals as compared to controls was revealed. Hence, the polymer is safe at the doses used within 6 months of injection.

Ceramidastin, a novel bacterial ceramidase inhibitor, produced by Penicillium sp. Mer-f17067.

Decrease of ceramide in the skin is one of the aggravating factors of atopic dermatitis. The skin is often infected by ceramidase-producing bacteria, such as Pseudomonas aeruginosa. The bacterial ceramidase then degrades ceramide in the skin. To develop anti-atopic dermatitis drugs, we searched for ceramidase inhibitors, which led to the discovery of ceramidastin, a novel inhibitor of bacterial ceramidase, from the culture broth of Penicillium sp. Mer-f17067. Ceramidastin inhibited the bacterial ceramidase with an IC(50) value of 6.25 microg ml(-1). Here we describe the isolation, physicochemical properties, structure determination and biological activity of ceramidastin.

A dimethylmaleic acid-melittin-polylysine conjugate with reduced toxicity, pH-triggered endosomolytic activity and enhanced gene transfer potential.

BACKGROUND: Poor endosomal release is one major barrier of gene delivery. Endosomolytic polyethylenimine-melittin conjugates have shown to enhance gene transfer efficiency; however, cytotoxicity due to their general membrane-destabilizing properties limits their application. To overcome this drawback we grafted a polycation with a masked pH-responsive melittin derivate and investigated lytic activity, gene transfer efficiency and cytotoxicity of the resulting conjugate. METHODS: Melittin (Mel) was modified with dimethylmaleic anhydride (DMMAn) and covalently coupled to poly-L-lysine (PLL). The membrane lytic activity was analyzed after incubation at neutral or endosomal pH. PLL-DMMAn-Mel polyplexes were generated in HEPES-buffered glucose and tested in transfection experiments using luciferase as reporter gene. Cellular cytotoxicity was analyzed by measurement of membrane integrity and metabolic activity. RESULTS: Covalent attachment of DMMAn-modified melittin to PLL resulted in a pH-responsive conjugate. No lytic activity was observed at neutral pH; after acidic cleavage of the protecting groups at pH 5 lytic activity was regained. Acute toxicity was greatly reduced (as compared to PLL-Mel or even unmodified PLL) and high gene expression levels (up to 1800-fold higher than unmodified PLL) were obtained. CONCLUSIONS: Modification of the polycationic carrier PLL with DMMAn-masked melittin not only enhances gene transfer efficiency, but also strongly reduces the acute toxicity of melittin and PLL. Hence this modification might be useful for optimizing polycationic gene carriers.

Preclinical toxicity study of a male injectable antifertility agent (styrene maleic anhydride) in rhesus monkeys, Macaca mulatta.

Polymer styrene maleic anhydride dissolved in dimethyl sulphoxide was injected intravasally to rhesus monkeys at the dose levels of 100 mg, 250 mg, and 500 mg in each vas deferens in low, high, and highest dose groups respectively, while control group received 0.5 ml DMSO in each vas deferens. The systemic toxicity study of the implant was carried out for 90 d. There had been no alteration in any of the toxicity parameters, ie, body weight, haematological, biochemical or histopathological, in the test animals as compared to control animals. Hence, it is concluded that the polymer SMA injected at above doses appears to be safe in our experiment.

A 6-month multispecies inhalation study with maleic anhydride.

This study was initiated to assess the safety of atmospheres containing maleic anhydride. Accordingly, rats (15/sex/group), hamsters (15/sex/group), and monkeys (3/sex/group) were treated 6 hr a day 5 days a week for 6 months. Atmospheres were generated by subliming maleic anhydride and were monitored using Tenax collection columns and gas chromatography to detect total maleic; i.e., maleic anhydride plus maleic acid. The mean analytical concentrations were 0, 1.1, 3.3, and 9.8 mg/m3 of total maleic. Dose-related signs of nasal and ocular irritation were observed at each test level in all three species; signs included discharge, sneezing, gasping, and coughing. No significant treatment-related mortality was observed in any species. While reduced weight gains were observed only in mid- and high-dose rats, their terminal body weights were greater than 90% of control values. No treatment-related effects were observed in hematology, clinical chemistry, urinalysis, and pulmonary function tests. Although microscopic evaluation of tissue revealed evidence of nasal irritation in all species, there was no evidence of systemic toxicity which was directly attributed to maleic anhydride. While the results of this study support the current ACGIH TLV and OSHA PEL of 1 mg/m3 regarding systemic toxicity, continuous exposure at this level during the day may produce some signs of irritation.

Teratology and multigeneration reproduction studies with maleic anhydride in rats.

These studies were initiated to evaluate the effects of maleic anhydride on development and reproduction in CD rats. In the teratology study, pregnant rats (19-23/group) received 0, 30, 90, or 140 mg/kg/day maleic anhydride in corn oil orally from Days 6-15 of gestation and fetuses were examined for gross soft tissue and skeletal defects. A reduced weight gain or weight loss was observed in all maleic anhydride-treated groups between Days 6 and 9; however, mean weights of all groups were within 5% of control on Days 15 and 20. No treatment-related effects on fetal development were observed. In the multigeneration study, rats (10 males and 20 females/group) received 0, 20, 55, or 150 mg/kg/day maleic anhydride in corn oil orally and were mated to produce two generations, each with two litters. Groups of the same size from the second litter were used for subsequent generations and were given the same dose of maleic anhydride as were their parents. The high-dose group was terminated during the second generation due to treatment-related mortality in adults. Renal cortical necrosis occurred in high-dose Fo males and females. Increased kidney weights were observed in low- and mid-dose adult F1 females. No treatment-related effects on reproduction were observed with maleic anhydride at doses up to 55 mg/kg/day over two generations.

Teratological evaluation of an injectable male antifertility agent, styrene maleic anhydride, in rats.

Polymer styrene Maleic anhydride (SMA) dissolved in dimethylsulphoxide (DMSO) was injected into the lumen in each vas deferens of male rats at dose levels of 0.25 mg, 0.50 mg, 1.0 mg, 2.5 mg, and 5.0 mg, while control rats (groups 1) received 0.03 mL DMSO in each vas deferens. After 4 weeks, the lumen was flushed with 0.1 mL DMSO and the animals were left for another 6 weeks to regain fertility. These rats were mated with virgin and coeval females. No anomaly was observed which could be related to teratogenic action of the polymer in pregnant mothers or fetuses.

Differential neutralizing effect of tiopronin on the toxicity of neocarzinostatin and SMANCS: a new rescue cancer chemotherapy.

The toxic effect and antitumor activity of neocarzinostatin (NCS) and SMANCS [copoly(styrenemaleic acid)-conjugated NCS] were greatly affected by N-(2-mercaptopropionyl)-glycine [tiopronin] both in vitro and in vivo, in cultured HeLa cells and RL male 1 tumor-bearing mice. The cytotoxicity of NCS and SMANCS against HeLa cells was remarkably reduced by the addition of tiopronin during drug treatment. Interestingly, the neutralizing effect of tiopronin on the toxicity of SMANCS was greater than that in the case of NCS. In the continuous presence of 10 mM tiopronin during a 1 h drug treatment, the 50% cell-killing doses of NCS and SMANCS were increased 72 and 208 times as compared to those without tiopronin, respectively, whereas tiopronin itself has no cytotoxicity to HeLa cells up to 100 mM. Furthermore, more effective reduction of the lethal toxicity of SMANCS was observed by the intraperitoneal (ip) administration of tiopronin after ip injection of a lethal dose of SMANCS as compared to the same protocol in the case of NCS in mice. Therapeutic studies on RL male 1 tumor-bearing mice revealed that delayed (time lag) ip administration of tiopronin after high-dose SMANCS administration ip was much superior to the combination of NCS with tiopronin, or SMANCS alone. In this time-lag combination chemotherapy of SMANCS with tiopronin, 60% of treated mice survived more than 60 days after tumor inoculation, while all the untreated control mice died within 20 days.

Combined effect of radiation and YM-881 (SMANCS) on murine tumors and bone marrow.

The combined effect of radiation and YM-881 (SMANCS) was studied in vitro and in vivo. When 0.25 microgram/ml of YM-881 was simultaneously combined with radiation, during and after irradiation for 30 min in total, Dq decreased from 3.3 Gy to 1.4 Gy without changing D0 in the dose-survival curve of exponentially growing SCC VII tumor cells. Five or ten times administrations of 0.1 mg/kg YM-881 at an interval of 24 h did not inhibit tumor growth. However, administration of 0.1 mg/kg YM-881 just before every irradiation which was repeated five times at an interval of 24 h yielded dose modifying factors (DMFs) of 1.8-1.2 when the tumor response to treatment was evaluated by the time for the tumors to regrow to three times the original volume. Administration of YM-881 ten times just before every irradiation yielded DMFs of 1.3-1.2. Adverse effects of the combination on bone marrow were examined by spleen colony assay. After five injections of 0.1 mg/kg YM-881, the mean number of CFU-S per femur decreased to 77% of the pretreatment level, but this was not significant statistically (0.1 greater than p greater than 0.05). The slope of radiation response curve for CFU-S per femur was not affected by the combination.

Interleukins 5 and 13 characterize immune responses to respiratory sensitizing acid anhydrides.

There is some debate regarding whether occupational asthma induced by respiratory sensitizing acid anhydrides is mediated by the induction of T helper (Th) 2-type responses and the production of IgE, with failure to detect specific IgE antibody in some symptomatic patients. In the current investigations, cytokine secretion profiles induced in draining lymph node cells (LNC) by topical application to BALB/c strain mice of trimellitic anhydride (TMA), phthalic anhydride (PA) and maleic anhydride (MA) have been examined. Responses were compared with those induced by exposure to 2,4-dinitrochlorobenzene (DNCB), a contact allergen that lacks respiratory sensitizing potential. Exposure to all three acid anhydrides stimulated vigorous expression of interleukin (IL)-5, IL-10 and IL-13 but relatively low levels of the type 1 cytokines interferon-gamma (IFN-gamma) and IL-12. In addition, TMA-activated LNC expressed high levels of mitogen-inducible IL-4 whereas MA and PA displayed a lesser potential to elaborate this cytokine. The DNCB-stimulated LNC exhibited the converse type 1 phenotype of cytokine expression. The CD4(+) Th2 cells were the primary source of type 2 cytokines. Respiratory sensitizing acid anhydrides induce a predominantly Th2 cytokine phenotype, including the expression of IL-5 and IL-13, cytokines which in the presence of only very low levels of IL-4 may provide for an IgE-independent mechanism for the development of chemical respiratory allergy. These data provide additional support for the use of cytokine secretion profiling for the prospective identification of chemical respiratory allergens.

Biological activity of maleic anhydride copolymers. I. Biocompatibility and antitumoural effects of maleic anhydride-vinyl acetate, maleic anhydride-methyl methacrylate and maleic anhydride-styrene copolymers.

A series of maleic anhydride (MA)-vinyl acetate (VA), MA-methyl methacrylate (MM), and MA-styrene (S) copolymers were prepared and characterized. On employing various amounts of initiator, maleic anhydride-vinyl acetate, methyl methacrylate, and styrene copolymers with molecular weights ranging between 18,000 and 200,000 have been obtained. The in vivo and in vitro tests performed on K562 cellular cultures (human chronic myeloid leukemia) have shown that, as a function of the molecular weight, the synthesized copolymers demonstrate a 50% in vitro cytotoxicity and an average tumour regression of maximum 68%.