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Thifluzamide is widely used fungicide and frequently detected in aquatic system. In this study, the toxicity of fungicide thifluzamide to non-targeted aquatic organisms was investigated for neuroendocrine disruption potentials. Here, zebrafish embryos were exposed to a series of concentrations of thifluzamide for 6 days. The results showed that both the development of embryos/larvae and the behavior of hatched larvae were significantly affected by thifluzamide. Importantly, the decreased activity of acetylcholinesterase (AchE) and the increased contents of neurotransmitters such as serotonin (5-HT) and norepinephrine (NE), along with transcriptional changes of nervous system related genes were observed following 4 days exposure to thifluzamide. Besides, the decreased contents of triiodothyronine (T3) and thyroxine (T4) in whole body, as well as significant expression alteration in hypothalamic-pituitary-thyroid (HPT) axis associated genes were discovered in zebrafish embryos after 4 days of exposure to thifluzamide. Our results clearly demonstrated that zebrafish embryos exposed to thifluzamide could disrupt neuroendocrine, compromise behavior and induce developmental abnormality, suggesting impact of this fungicide on developmental programming in zebrafish., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Co-occurrence of pesticide residues in food commodities raises a potential safety issue as their mixture effects on human health are largely unknown. In a previous study, we reported the toxicological effects (pathology and histopathology) of imazalil (IMZ), thiacloprid (THI), and clothianidin (CTD) alone and in binary mixtures in a 28-day oral gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg body weight/day) ranging from a typical toxicological reference value to a clear effect dose were applied. In the present study, we undertook a transcriptomics analysis of rat livers by means of total RNA sequencing (RNA-Seq). Bioinformatic data analysis involving Ingenuity Pathway Analysis (IPA) was used to gain mechanistic information on hepatotoxicity-related pathways affected after treatment with the pesticides, alone and in mixtures. Our data show that 2986 genes were differentially regulated by CTD while IMZ and THI had effects on 194 and 225 genes, respectively. All three individual compounds shared a common subset of genes whose network is associated with xenobiotic metabolism and nuclear receptor activation. Similar networks were retrieved for the mixtures. Alterations in the expression of individual genes were in line with the assumption of dose addition. Our results bring new insight into the hepatotoxicity mechanisms of IMZ, THI, and CTD and their mixtures.

Chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT) are biocidal preservatives and the active ingredients in Kathon CG, which contains ca. 1.5% mixture of CMIT and MIT at a ratio of 3:1 (CMIT/MIT). CMIT/MIT was misused as humidifier disinfectant products, which caused serious health problems in Korea. Here, the vascular effects of CMIT/MIT were investigated to evaluate claims of putative cardiovascular toxicity observed in humidifier disinfectant users. CMIT/MIT did not affect the basal tension of the rat thoracic aorta up to 2.5 μg/mL in myograph experiments. Instead, pretreatment with CMIT/MIT impaired phenylephrine- or 5-hydroxytryptamine-induced vasoconstriction in a range of 0.5-2.5 μg/mL, which was largely irreversible and not recovered by washing out the CMIT/MIT. Similarly, the application of CMIT/MIT to pre-contracted aorta caused a gradual loss of tension. In primary cultured vascular smooth muscle cells (VSMCs), CMIT/MIT caused thiol depletion, which in turn led to cytosolic Zn 2+ elevation and reactive oxygen species (ROS) formation. CMIT/MIT-induced shrinkage, detachment, and lysis of VSMCs depending on the concentration and the treatment time. All events induced by CMIT/MIT were prevented by a thiol donor N-acetylcysteine (NAC). Cytolysis could be inhibited by a Zn 2+ chelator TPEN and a superoxide scavenger TEMPOL, whereas they did not affect shrinkage and detachment. In accordance with these results, CMIT/MIT-exposed aortas exhibited dissociation and collapse of tissue in histology analysis. Taken together, CMIT/MIT causes functional impairment and tissue damage to blood vessels by depleting thiol and thereby elevating cytosolic Zn 2+ and generating ROS. Therefore, exposure to CMIT/MIT in consumer products may be a risk factor for cardiovascular disorders.

Cyclin-dependent kinases (CDKs) are a family of kinases associated predominantly with cell cycle control, making CDK inhibitors interesting candidates for anti-cancer therapeutics. However, retinal toxicity (loss of photoreceptors) has been associated with CDK inhibitors, including the pan-CDK inhibitor AG-012896. The purpose of this research was to use a novel planar sectioning technique to determine CDK expression profiles in the ex vivo human retina with the aim of identifying isoforms responsible for CDK retinotoxicity. Four CDK isoforms (CDK11, 16, 17 and 18) were selected as a result of IC 50 data comparing neurotoxic (AG-012986 and NVP-1) and non-neurotoxic (dinaciclib and NVP-2) CDK inhibitors, with IC 50 s at CDK11 showing a clear difference between the neurotoxic and non-neurotoxic drugs. CDK11 was maximally expressed in the photoreceptor layer, whereas CDK16, 17 and 18 showed maximal expression in the inner nuclear layer. CDK5 (an isoform associated with retinal homeostasis) was maximally expressed in the retinal ganglion cell layer. Apart from CDK18, each isoform showed expression in the photoreceptor layer. The human Müller cell line MIO-M1 expressed CDK5, 11, 16 and 17 and AG-01298 (0.02-60 µM) caused a dose-dependent increase in MIO-M1 cell death. In conclusion, CDK11 appears the most likely candidate for mediation of photoreceptor toxicity. RNA profiling can be used to determine the distribution of genes of interest in relation to retinal toxicity in the human retina.

In nearly every species examined, administration of the persistent environmental pollutant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD) causes profound immune suppression and thymic atrophy in an aryl hydrocarbon receptor (AhR) dependent manner. Moreover, TCDD alters the development and differentiation of thymocytes, resulting in decreases in the relative proportion and absolute number of double positive (DP, CD4 + CD8 + ) thymocytes, as well as a relative enrichment in the relative proportion and absolute number of double negative (DN, CD4 - CD8 - ) and single-positive (SP) CD4 + CD8 - and CD4 - CD8 + thymocytes. Previous studies suggested that the target for TCDD-induced thymic atrophy resides within the hemopoietic compartment and implicated apoptosis, proliferation arrest of thymic progenitors, and emigration of DN thymocytes to the periphery as potential contributors to TCDD-induced thymic atrophy. However, the precise cellular and molecular mechanisms involved remain largely unknown. Our results show that administration of 10 µg/kg TCDD and 8 mg/kg 2-(1H-indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE) induced AhR-dependent thymic atrophy in mice on day 7, whereas 100 mg/kg indole 3-carbinol (I3C) did not. Though our studies demonstrate that TCDD triggers a twofold increase in the frequency of apoptotic thymocytes, TCDD-induced thymic atrophy is not dependent on Fas-FasL interactions, and thus, enhanced apoptosis is unlikely to be a major mechanistic contributor. Finally, our results show that activation of the AhR in CD11c + dendritic cells is directly responsible for TCDD-induced alterations in the development and differentiation of thymocytes, which results in thymic atrophy. Collectively, these results suggest that CD11c + dendritic cells play a critical role in mediating TCDD-induced thymic atrophy and disruption of T lymphocyte development and differentiation in the thymus.

BAL30072 is a new monocyclic β-lactam antibiotic under development which provides a therapeutic option for the treatment of severe infections caused by multi-drug-resistant Gram-negative bacteria. Despite the absence of liver toxicity in preclinical studies in rats and marmosets and in single dose clinical studies in humans, increased transaminase activities were observed in healthy subjects in multiple-dose clinical studies. We, therefore, initiated a comprehensive program to find out the mechanisms leading to hepatocellular injury using HepG2 cells (human hepatocellular carcinoma cell line), HepaRG cells (inducible hepatocytes derived from a human hepatic progenitor cell line), and human liver microtissue preparations. Our investigations demonstrated a concentration- and time-dependent reduction of the ATP content of BAL30072-treated HepG2 cells and liver microtissues. BAL30072 impaired oxygen consumption by HepG2 cells at clinically relevant concentrations, inhibited complexes II and III of the mitochondrial electron transport chain, increased the production of reactive oxygen species (ROS), and reduced the mitochondrial membrane potential. Furthermore, BAL 30072 impaired mitochondrial fatty acid metabolism, inhibited glycolysis, and was associated with hepatocyte apoptosis. Co-administration of N-acetyl-L-cysteine partially protected hepatocytes from BAL30072-mediated toxicity, underscoring the role of oxidative damage in the observed hepatocellular toxicity. In conclusion, BAL30072 is toxic for liver mitochondria and inhibits glycolysis at clinically relevant concentrations. Impaired hepatic mitochondrial function and inhibition of glycolysis can explain liver injury observed in human subjects receiving long-term treatment with this compound.

Purpose: The aim of the study is to investigate risk factors for sensitization to preservatives and to examine to which extent different preservatives are registered in chemical products for occupational use in Denmark., Methods: A retrospective epidemiological observational analysis of data from a university hospital was conducted. All patients had occupational contact dermatitis and were consecutively patch tested with 11 preservatives from the European baseline series and extended patch test series during a 5-year period: 2009-2013. Information regarding the same preservatives in chemical products for occupational use ('substances and materials') registered in the Danish Product Register Database (PROBAS) was obtained., Results: The frequency of preservative contact allergy was 14.2% (n = 141) in 995 patients with occupational contact dermatitis. Patients with preservative contact allergy had significantly more frequently facial dermatitis (19.9 versus 13.1%) and age > 40 years (71.6 versus 45.8%) than patients without preservative contact allergy, whereas atopic dermatitis was less frequently observed (12.1 versus 19.8%). Preservative contact allergy was more frequent in painters with occupational contact dermatitis as compared to non-painters with occupational contact dermatitis (p < 0.001). This was mainly caused by contact allergy to methylisothiazolinone and contact allergy to formaldehyde. Analysis of the registered substances and materials in PROBAS revealed that preservatives occurred in several product categories, e.g., 'paints and varnishes', 'cleaning agents', 'cooling agents', and 'polishing agents'. Formaldehyde and isothiazolinones were extensively registered in PROBAS., Conclusions: The extensive use of formaldehyde and isothiazolinones in chemical products for occupational use may be problematic for the worker. Appropriate legislation, substitution, and employee education should be prioritized.

Parabens, methylisothiazolinone (MI) and its derivative methylchloroisothiazolinone (MCI), are commonly used as preservatives in personal care products. They can cause hypersensitivity reactions of the human skin. We have tested a set of nine parabens, MI alone and in combination with MCI in the loose-fit coculture-based sensitization assay (LCSA). The coculture of primary human keratinocytes and allogenic dendritic cell-related cells (DC-rc) in this assay emulates the in vivo situation of the human skin. Sensitization potency of the test substances was assessed by flow cytometric analysis of the DC-rc maturation marker CD86. Determination of the concentration required to cause a half-maximal increase in CD86-expression (EC50sens) allowed a quantitative evaluation. The cytotoxicity of test substances as indicator for irritative potency was measured by 7-AAD (7-amino-actinomycin D) staining. Parabens exhibited weak (methyl-, ethyl-, propyl- and isopropylparaben) or strong (butyl-, isobutyl-, pentyl- and benzylparaben) effects, whereas phenylparaben was found to be a moderate sensitizer. Sensitization potencies of parabens correlated with side chain length. Due to a pronounced cytotoxicity, we could not estimate an EC50sens value for MI, whereas MI/MCI was classified as sensitizer and also showed cytotoxic effects. Parabens showed no (methyl- and ethylparaben) or weak irritative potencies (propyl-, isopropyl-, butyl-, isobutyl-, phenyl- and benzylparaben), only pentylparaben was rated to be irritative. Overall, we were able to demonstrate and compare the sensitizing potencies of parabens in this in vitro test. Furthermore, we showed an irritative potency for most of the preservatives. The data further support the usefulness of the LCSA for comparison of the sensitizing potencies of xenobiotics.

The carboxylic acid NSAID fenclozic acid exhibited an excellent preclinical safety profile and promising clinical efficacy, yet was withdrawn from clinical development in 1971 due to hepatotoxicity observed in clinical trials. A variety of modern in vitro approaches have been used to explore potential underlying mechanisms. Covalent binding studies were undertaken with [(14)C]-fenclozic acid to investigate the possible role of reactive metabolites. Time-dependent covalent binding to protein was observed in NADPH-supplemented liver microsomes, although no metabolites were detected in these incubations or in reactive metabolite trapping experiments. In human hepatocytes, covalent binding was observed at lower levels than in microsomes and a minor uncharacterizable metabolite was also observed. In addition, covalent binding was observed in incubations undertaken with dog and rat hepatocytes, where a taurine conjugate of the drug was detected. Although an acyl glucuronide metabolite was detected when liver microsomes from human, rat and dog were supplemented with UDPGA, there was no detectable UDPGA-dependent covalent binding. No effects were observed when fenclozic acid was assessed for P450-dependent and P450-independent cytotoxicity to THLE cell lines, time-dependent inhibition of five major human cytochrome P450 enzymes, inhibition of the biliary efflux transporters BSEP and MRP2 or mitochondrial toxicity to THLE or HepG2 cells. These data suggest that Phase 1 bioactivation plays a role in the hepatotoxicity of fenclozic acid and highlight the unique insight into mechanisms of human drug toxicity that can be provided by investigations of biotransformation and covalent binding to proteins.

In the understanding of colony loss phenomena, a worldwide crisis of honeybee colonies which has serious consequences for both apiculture and bee-pollination-dependent farm production, analytical chemistry can play an important role. For instance, rapid and accurate analytical procedures are currently required to better assess the effects of neonicotinoid insecticides on honeybee health. Since their introduction in agriculture, neonicotinoid insecticides have been blamed for being highly toxic to honeybees, possibly at the nanogram per bee level or lower. As a consequence, most of the analytical methods recently optimized have focused on the analysis of ultratraces of neonicotinoids using liquid chromatography-mass spectrometry techniques to study the effects of sublethal doses. However, recent evidences on two novel routes-seedling guttations and seed coating particulate, both associated with corn crops-that may expose honeybees to huge amounts of neonicotinoids in the field, with instantly lethal effects, suggest that selected procedures need optimizing. In the present work, a simplified ultra-high-performance liquid chromatography-diode-array detection method for the determination of neonicotinoids in single bees has been optimized and validated. The method ensures good selectivity, good accuracy, and adequate detection limits, which make it suitable for the purpose, while maintaining its ability to evaluate exposure variability of individual bees. It has been successfully applied to the analysis of bees in free flight over an experimental sowing field, with the bees therefore being exposed to seed coating particulate released by the pneumatic drilling machine.

Biocides are added to biodiesels to inhibit and remove microbial growth. The effects of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), a candidate biodiesel biocide, were studied using freshly isolated rat alveolar macrophages (AM) and NR8383 cell line. CMIT markedly inhibited phagocytic oxidative burst as measured by zymosan-induced chemiluminescence, and cellular cytokine secretion as measured by zymosan-induced TNF-α secretion. The 50% inhibition concentration (LC(50)) for CMIT was 0.002-0.004 mM for both cellular functions. AM exposed to CMIT for as little as 2 min showed markedly inhibited functions that persisted for at least 5 h. Sodium metabisulfite was able to partially neutralize the inhibitory activity of CMIT. Cysteine and glutathione, when present at a molar ratio of 2-1 or higher against CMIT, were effective neutralizers, while serine, histidine, alanine, and albumin were without effect. When the AM testing system was used to compare the toxicity of CMIT against three other candidate biodiesel biocides, methylene dithiocyanate (MDC) was found to be of comparable toxicity to CMIT, 2-methyl-4-isothiazolin-3-one (MIT) was much less toxic, and dimethyl acetylenedicarboxylate (DMAD) was non-toxic. Because AM is among the first cell-type exposed to inhaled biodiesel aerosols, the result suggested that CMIT present in biodiesel may produce respiratory effects, and further investigations including animal studies are warranted.

In September 2008, organotin (Ot) compounds were prohibited from being used worldwide. From 1997 onward in Japan, the production of paints containing TBT (tributylin) compounds was prohibited, and thus alternatives to Ot antifoulants have been used since then. It has been said that the decomposition characteristics of these materials are better than those of Ot compounds. The toxicity of alternative Ot antifoulants (e.g., diuron, irgarol 1051®, and Sea-Nine 211®) and Ot compounds (TBT and TPT (triphenyltin)), using oysters that inhabit a large area of Hiroshima Bay, were evaluated. The results showed that the toxicity of diuron and irgarol 1051 is very low, and the toxicity of Sea-Nine 211 is almost the same as that of TPT. Sea-Nine 211's effect was stronger on oysters than other shellfish, causing concern about the extent of Sea-Nine 211's impact on oyster development.

Acetamiprid and thiamethoxam are insecticides introduced for pest control, but they can also affect non-target insects such as honeybees. In insects, these neonicotinoid insecticides are known to act on acetylcholine nicotinic receptors but the behavioral effects of low doses are not yet fully understood. The effects of acetamiprid and thiamethoxam were studied after acute sublethal treatment on the behavior of the honeybee (Apis mellifera) under controlled laboratory conditions. The drugs were either administered orally or applied topically on the thorax. After oral consumption acetamiprid increased sensitivity to antennal stimulation by sucrose solutions at doses of 1 microg/bee and impaired long-term retention of olfactory learning at the dose of 0.1 microg/bee. Acetamiprid thoracic application induced no effect in these behavioral assays but increased locomotor activity (0.1 and 0.5 microg/bee) and water-induced proboscis extension reflex (0.1, 0.5, and 1 microg/bee). Unlike acetamiprid, thiamethoxam had no effect on bees' behavior under the conditions used. Our results suggest a particular vulnerability of honeybee behavior to sublethal doses of acetamiprid.

The toxicity of three antifoulants (Sea-Nine, Irgarol, and TBT) was determined individually and in mixtures in two tests with microalgae. Effects on periphyton community photosynthesis and reproduction of the unicellular green algae Scenedesmus vacuolatus were investigated. The tested antifoulants were highly toxic in both tests. Observed mixture toxicities were compared with predictions derived from two concepts: Independent Action (IA), assumed to be more relevant for the tested mixtures that were composed of dissimilarly acting substances, and Concentration Addition (CA), regarded as a reasonable worst-case approach in predictive mixture hazard assessment. Despite the corresponding mechanistic basis, IA failed to provide accurate predictions of the observed mixture toxicities. Results show the same pattern in both assays. Mixture effects at high concentrations were slightly overestimated and effects at low concentrations were slightly underestimated. Maximum observed deviations between observed and IA-predicted concentrations amount to a factor of 4. The suggested worst-case approach using CA was protective only in effect regions above 20%. Nevertheless, the application of any concept that accounts for possible mixture effects is more realistic than the present chemical-by-chemical assessment.

2-(Thiocyanomethylthio)benzothiazole (TCMTB) is a biocide used in the leather, pulp and paper, and water-treatment industries. TCMTB may enter aquatic ecosystems during its manufacture and use. TCMTB is environmentally unstable; therefore, it is important to evaluate the toxicity of the more persistent degradation products. This study compared the toxicity of TCMTB with its degradation products 2-mercaptobenzothiazole (2-MBT), 2-(methylthio)benzothiazole (MTBT), benzothiazole (BT), and 2-hydroxybenzothiazole (HOBT). Toxicity was determined using Ceriodaphnia dubia 48-hour acute and 7-day chronic test protocols. TCMTB was the most toxic compound evaluated in both the acute and chronic tests with EC50s of 15.3 and 9.64 microg/L, respectively. 2-MBT, the first degradation product, was the second most toxic compound with acute and chronic EC50s of 4.19 and 1.25 mg/L, respectively. The toxicity of MTBT and HOBT were similar with acute EC50s of 12.7 and 15.1 mg/L and chronic EC50s of 6.36 and 8.31 mg/L, respectively. The least toxic compound was BT with acute and chronic EC50s of 24.6 and 54.9 mg/L, respectively. TCMTB was orders of magnitude more toxic than its degradation products. Toxicity data on these benzothiazole degradation products is important because of concerns regarding their release, degradation, persistence, and non-target organism effects in aquatic ecosystems.

Rationale: Modulation of metabotropic glutamate receptor (mGluR) subtypes represents a novel approach for the treatment of neurological and psychiatric disorders., Objectives: This study was conducted to investigate the role of the mGluR5 and mGluR1 subtypes in the modulation of pain and anxiety., Methods: The mGluR5 antagonists, 2-methyl-6-(phenylethynyl)pyridine (MPEP) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP), and the mGluR1 antagonist, (4-methoxy-phenyl)-(6-methoxy-quinazolin-4-yl)-amine HCl (LY456236), were tested in models of pain [mouse formalin test, rat spinal nerve ligation (SNL)] and anxiety [Vogel conflict, conditioned lick suppression (CLS)], and their efficacious effects were compared to any associated side effects., Results: The systemic administration of MPEP, MTEP, and LY456236 reduced hyperalgesia induced by formalin and mechanical allodynia following SNL. However, only LY456236 completely reversed the allodynia. In the anxiety models, MPEP (3--30 mg/kg), MTEP (3--10 mg/kg), and LY456236 (10--30 mg/kg) produced anxiolytic-like effects similar to the benzodiazepine, chlordiazepoxide (CDP, 6 mg/kg). However, only MPEP and MTEP were able to produce a level of anxiolysis comparable to CDP. In a series of tests examining potential side effects, MPEP and MTEP reduced body temperature and locomotor activity and impaired operant responding for food and rotarod performance at doses of 3--30 and 1--30 mg/kg, respectively. LY456236 reduced operant responding at 30 mg/kg., Conclusion: Both mGluR5 and mGluR1 antagonists are effective in models of pain and anxiety. However, an mGluR1 antagonist was more efficacious than the two mGluR5 antagonists in the pain models, which, conversely, appeared more efficacious in the anxiety models. These findings support the potential utility of mGluR5 and mGluR1 antagonists for both the treatment of chronic pain and as novel anxiolytics.

Thiazolidinediones (TZDs) are effective for the treatment of adult-onset insulin-resistant diabetes. Unfortunately, TZDs are associated with sporadic hepatic dysfunction that is not predictable from experimental animal studies. We investigated the response of isolated rat and human hepatocytes to various TZDs using biochemical assays, coherent multiprobe fluorescence microscopy and flow cytometric analyses. The results identified direct effects of TZD on mitochondria from live human and rodent hepatocytes. The multiprobe fluorescence assays showed disruption of mitochondrial activity as an initiating event followed by increased membrane permeability, calcium influx and nuclear condensation. Other TZD-related cellular effects were increased hepatic enzyme leakage, decreased reductive metabolism and cytoplasmic adenosine triphosphate depletion. Mitochondrial effects were similar in cryopreserved hepatocytes from diabetic or non-diabetic donors. Peripheral blood mononuclear cells (PBMCs) had baseline mitochondrial energetics and metabolism comparable with isolated hepatocytes. Mitochondrial effects in isolated hepatocytes were found in human PBMCs exposed to the TZDs. The relative potency of TZDs for causing hepatocyte and PBMC effects was troglitazone > pioglitazone > rosiglitazone. These studies clearly demonstrated that hepatic alterations in vitro are characteristic of TZDs, with only quantitative differences in subcellular organelle dysfunction. Monitoring mitochondrial function in isolated PBMCs may be beneficial in diabetics undergoing TZD therapy.

FK506 and cyclosporin A (CsA) are two potent immunosupressants with similar toxicity profile. Nephrotoxicity is the main adverse effect of both compounds. The aim of this study is to compare the in vitro nephrotoxic effects on renal epithelial cell line LLC-PK1 by measuring cell viability and energy status as evaluated by concentrations of ATP and ATP metabolites. Cell viability (expressed as IC50 was assessed via thiazolyl blue (MTT) assay after incubation for 4-24 h with FK506 or CsA. ATP and its metabolites were determined by HPLC after 4 and 6 h incubation with FK506 or CsA alone at the respective IC50. Both FK506 and CsA decreased cell viability to similar extents, in a dose- and time-dependent manner. After 4 h incubation, both drugs decreased ATP levels (-25%) and increased uric acid levels. However, the latter percentage increase was twofold higher with CsA (18%) than with FK506 (9%). The energy charge, calculated according to levels of adenine nucleotides, was decreased by 10% in FK506-treated cells and by 27% in CsA-treated cells. At the end of 6-h incubation, FK506-treated cells maintained ATP levels coupled with energy charge at near control levels whereas the levels were 32% lower in CsA treated cells. Compared to the 4 h-incubation, the increase in uric acid was similar for FK506 but was doubled with CsA. The decrease in cell integrity and ATP depletion induced by CsA in LLC-PK1 cells was only transiently observed with FK506. By preserving energy status, FK506 leads to fewer metabolic disturbances than CsA in the renal epithelial cell line LLC-PK1, demonstrating a minor potential nephrotoxicity.

Aldophosphamide thiazolidine (NSC 613060) and aldophosphamide perhydrothiazine (NSC 612567), which hydrolyse spontaneously to 4-hydroxycyclophosphamide (4-OH-CP) in aqueous solution, were synthesised. These substances are prototypes of a new class of prodrugs for activated oxazaphosphorines. They were developed according to our hypothesis on the mechanism of action of oxazaphosphorine cytostatics. According to this hypothesis, toxicity and canceroselectivity are the results of phosphoramide mustard (PAM) release from 4-OH-CP catalysed by two classes of phosphodiesterase. 4-OH-CP toxicity results (a) from oxazaphosphorine-specific toxicity due to reactivity of the hemiaminal group with thiol groups of membrane proteins and (b) from PAM release catalysed by ubiquitous phosphodiesterases present in blood and tissues. Specific cytotoxicity suitable for antitumour therapy is based on specific PAM release in the vicinity of the target molecule DNA by the exonuclease subsites of DNA polymerases delta and epsilon. To unfold this specific core, which, we assume, improves efficacy in cancer treatment, low, long-lasting concentrations of OH-CP have to be guaranteed beneath the affinity range of the ubiquitous phosphodiesterase. This goal is facilitated by the rapid transfer of 4-OH-CP released from the perhyrothiazine derivative NSC 612567 to protein SH groups, as shown by protein-binding studies. Half-lives of hydrolysis and dissociation constants of the thiazolidine and perhydrothiazine derivatives, in which the reactivity of the hemiaminal group is inactivated by inclusion into the thiazolidine or perhydrothiazine ring, were determined to be 23 h and 6.0 x 10(-6) mol/l for NSC 613060 and 1.5 h and 1.1 x 10(-4) mol/l for NSC 312567. Accordingly the compounds guarantee low but long-lasting steady-state concentrations of 4-OH-CP. The acute toxicity determined in mice was 2400 mg/kg for NSC 613060 and 1900 mg/kg for NSC 612567. Except for a 30% decrease in leucocytes, daily i.p. injections of 260 mg/kg NSC 612567 (15% of LD50) were tolerated without signs of toxicity over a period of 4 weeks. In contrast, equitoxic doses of cyclophosphamide caused severe signs of toxicity, only five daily applications were tolerated. In mice treated repeatedly with NSC 613060, oxazaphosphorine toxicity was overlapped by thiazolidine toxicity. Scheduled activity tests in mice bearing P815 ascites tumour showed optimal therapeutic response when mice were treated daily. Repeated applications of 4% LD50 of NSC 613060 and 13% LD50 of NSC 612567 prevented tumour growth in mice with advanced, P388 lymphomas, implanted subcutaneously, without signs of overall toxicity to the host.

In this report we describe the antiviral activity of 5,5'-diphenyl-3,3'-diisothiazole disulfide (DID) and discuss its mode of action. DID selectively inhibits the replication of poliovirus type 1 (therapeutic index = 255) by affecting some early process of the virus growth cycle. The compound does not interfere with adsorption and internalization of virus to HEp-2 cells, nor with uncoating of the viral RNA. However, no viral RNA synthesis occurs after 2 h post-infection in the presence of 50 microM DID. Thus, we investigated some molecular events in poliovirus replication occurring between uncoating and viral RNA synthesis. In our experimental design, we studied the activity of RNA polymerase complex isolated from HEp-2 infected cells in the presence or absence of DID. Our results showed that the RNA polymerase complex was formed in the presence of DID. On the contrary, DID markedly inhibited poliovirus RNA synthesis in a cell-free system using RNA polymerase complex isolated from infected cells. These findings indicate that DID may exert its antiviral activity by preventing viral RNA chain elongation via the inhibition of replicase activity and/or interfering with viral RNA polymerase complex.

30 female Wistar rats weighing between 70 and 90 g were fed for 8 months with 0,188% N-[4-(5-nitro-2-furyl)-2-thiazolyl] Formamide (FANFT). After this period a papillary tumor of the urinary bladder was demonstrable in each animal. Histological examination always revealed a transitional cell carcinoma with prevailing medium tumor stages; no distant metastases were found. The grade of malignancy was classified as medium and high. No significant statistical relationship could be ascertained between tumor stage and grade or between grade and weight, but a correlation was established between tumor stage and weight. After 12 months, 36/60 kidneys were found to be normal, whereas in 20/60 dysplasias and in 2/60 transitional cell carcinomas of the renal pelvis were observed; secondary hydronephrosis due to bladder tumors occured 4 times. Other organ changes were not noticeable.

After feeding with 0.188% N-[4-(5-nitro-2 furyl)-2-thiazolyl] formamide (FANFT), transitional cell carcinoma of the urinary bladder was induced in all female Wistar rats tested. Histological changes of the urothelium consisted of various degrees of hyperplasia and dysplasia. An infiltrating transitional cell carcinoma first appeared after 8 months. These results are compared with the findings of other authors, and divergencies of the tumour induction rates are discussed with respect to strain, sex and weight of experimental animals as well as concentration and amount of ingested carcinogen. Electron microscopy shows microvillous transformation of the luminal plasma membrane and appearance of a thick fluffy cell coat (glycocalyx). These changes are explained by an altered function of the Golgi complex occurring during malignancy and leading to a loss of the specific discoid vesicles of the urothelial cells.