New free-exchange model of EmrE transport.

EmrE is a small multidrug resistance transporter found in Escherichia coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a free-exchange model for EmrE antiport that is consistent with these results and recapitulates ∆pH-driven concentrative drug uptake. Kinetic modeling suggests that free exchange by EmrE sacrifices coupling efficiency but boosts initial transport speed and drug release rate, which may facilitate efficient multidrug efflux.

Comparative dermal toxicity of dicyclohexylcarbodiimide and diisopropylcarbodiimide in rodents.

Dicyclohexylcarbodiimide (DCC) and Diisopropylcarbodiimide (DIC) are two representative chemicals in the carbodiimide class of chemicals used in industry as stabilizing agents. There is a potential of dermal exposure to these agents in chemical, pharmaceutical and recombinant DNA industries. The National Toxicology Program conducted a number of animal studies to characterize toxicity and carcinogenicity of DIC and DCC. Dermal administration of DCC and DIC in F344/N rats and B6C3F1 mice for 90-days induced skin irritation at the site of application in a dose-dependent manner. Microscopically, dose-dependent increases in epidermal hyperplasia and chronic inflammation were observed. We further evaluated the effects of dermal exposure of DCC and DIC in p53 haploinsufficient and Tg.AC mouse models. Results revealed the skin as the primary target of DCC and DIC exposure as indicated by dose - dependent skin lesions (hyperplasia, inflammation and necrosis). DCC induced squamous cell papillomas in Tg.AC mice but did not induce any neoplastic lesions in p53 haploinsufficient mice. Dermal application of DIC did not induce any neoplastic lesions in Tg.AC mice and p53 haploinsufficient mice. Based on these studies, it was predicted that DIC would be negative and DCC positive for carcinogenic activity in the traditional two-year bioassay. In the subsequent studies, the carcinogenic potential of DIC only in F344 rats and B6C3F1 mice in a traditional 2-year chronic carcinogenicity bioassay was evaluated by the dermal route. Findings revealed the skin as the major target organ of toxicity in both sexes in rats and in male mice. There were no neoplastic lesions observed in rats or mice with the administration of DIC. In rats, there were clinical signs of toxicity in the highest dose-group which included ataxia, excitability, impaired gait, low muscle tone, abnormal breathing, lethargy, and seizures. This was accompanied by non-neoplastic lesions in the brain and lung only at the highest dose level. In conclusion, both DIC and DCC are dermal toxicants. DIC did not have any carcinogenic activity in transgenic mouse models or in the traditional NTP two-year carcinogenicity studies in F344 rats and B6C3F1 mice. DCC was positive in the Tg.AC mouse model and likely to be carcinogenic in the 2-year bioassay as well.

Multi-site TBT binding skews the inhibition of oligomycin on the mitochondrial Mg-ATPase in Mytilus galloprovincialis.

Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F(1)F(0)-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 µM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F(0), namely oligomycin and N,N'-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F(1), maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 µM TBT concentrations, a structural detachment of the two F(1) and F(0) domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 µM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F(0) proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F(1)F(0)-ATPase oligomycin sensitivity loss.

Isolation and characterization of a N,N'-dicyclohexylcarbodiimide-resistant mutant of Methanothermobacter thermautotrophicus with alterations to the ATP synthesis machinery.

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant toward the ATP-synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) was isolated. DCCD normally inhibits methanogenic electron-transport-driven ATP synthesis, however, the DCCD-resistant strain exhibited methanogenesis in the presence of 300 micromol/L DCCD. Total ATP synthesis was shown to be higher in the mutant strain, both in the presence and absence of DCCD. These results suggested a modification in the ATP-synthesizing system of the mutant strain. Using Blue Native PAGE combined with MALDI TOF/TOF mass spectrometry, increased concentrations of both the A(1) and A(o) subcomplexes of the A(1)A(o)-type synthase were identified in the mutant strain. However, no alterations were found in the structural genes (atp) for the A(1)A(o) ATP synthase. The results imply that DCCD resistance is a consequence of increased A(1)A(o) ATP synthase expression, and suggest that genes involved in regulating synthase expression are responsible for DCCD resistance.

NTP report on the toxicology studies of dicyclohexylcarbodiimide (CAS No. 538-75-0) in F344/N rats, B6C3F 1 mice, and genetically modified (FVB Tg.AC hemizygous) mice and carcinogenicity study of dicyclohexylcarbodiimide in genetically modified [B6.129-Trp53 tm1Brd (N5) haploinsufficient] mice (dermal studies).

Dicyclohexylcarbodiimide is used in industry as a stabilizing agent, coupling agent, and condensing agent. Its widespread use during protein synthesis in the recombinant DNA industry and in the synthesis of polypeptides in the chemical and pharmaceutical industries provides an increasing potential for low-level human exposure. Dicyclohexylcarbodiimide was nominated for study by The National Cancer Institute as a key representative of the carbodiimide chemical class because of its acute toxicity and the absence of data on potential health effects. Male and female F344/N rats and B6C3F 1 mice were administered dicyclohexylcarbodiimide (greater than 98% pure) dermally for 3 or 13 weeks. Female Tg.AC hemizygous and p53 haploinsufficient mice were administered dicyclohexylcarbodiimide dermally for 20 or 27 weeks, respectively. Genetic toxicology studies were conducted in Salmonella typhimurium, male F344/N rat bone marrow cells, and B6C3F 1 mouse peripheral blood erythrocytes. 3-WEEK STUDY IN F344/N RATS Groups of five male and five female rats were dermally administered 0.3 mL ethanol containing 0, 0.6, 1.8, 5.1, 15, or 45 mg dicyclohexylcarbodiimide, 5 days per week for 3 weeks. All males and females in the 15 and 45 mg groups, four 5.1 mg males, and all 5.1 mg females died before the end of the study. Of the surviving groups, final mean body weights were similar to those of the vehicle controls, although the one surviving 5.1 mg male rat lost weight during the study. Histopathologic examination of rats dosed with 5.1 mg dicyclohexylcarbodiimide or less revealed treatment-related lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, or chronic active inflammation in the dermis. 3-WEEK STUDY IN B6C3F 1 MICE Groups of five male and five female mice were dermally administered 0.1 mL of ethanol containing 0, 0.2, 0.6, 1.7, 5, or 15 mg dicyclohexylcarbodiimide, 5 days per week for 3 weeks. One 0.6 mg female mouse and all mice in the 1.7, 5, and 15 mg groups died before the end of the study. Final mean body weights of the 0.6 mg groups were significantly less than those of the vehicle controls, and animals in these groups generally lost weight during the study. Histopathologic examination of mice dosed with 1.7 mg dicyclohexylcarbodiimide or less revealed treatment-related lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, and acute or chronic active dermal inflammation. 13-WEEK STUDY IN F344/N RATS Groups of 10 male and 10 female core study rats were dermally administered 0, 0.75, 1.5, 3, 6, or 12 mg dicyclohexylcarbodiimide/kg body weight in ethanol, 5 days per week for 13 weeks; groups of 10 male and 10 female clinical pathology study rats were administered the same doses for 22 days. All 12 mg/kg male and female core study rats died or were found moribund and sacrificed prior to day 45. Final mean body weight and body weight gain of 6 mg/kg males were significantly less than those of the vehicle controls. The predominant clinical pathology changes suggest a secondary, treatment-related inflammatory leukogram and minimal decreased erythron of chronic inflammation that would be consistent with necrosis and chronic active inflammation of the skin. Significantly increased incidences of skin lesions at the site of application included epidermal hyperplasia in 3 mg/kg or greater males and 1.5 mg/kg or greater females, chronic active inflammation in 6 and 12 mg/kg males and 1.5 mg/kg or greater females, and epidermal necrosis in 12 mg/kg males. The incidences and severities of epidermal hyperplasia increased in a dose-related manner in both sexes of rats 13-WEEK STUDY IN B6C3F 1 MICE Groups of 10 male and 10 female mice were dermally administered 0, 1.5, 3, 6, 12, or 24 mg dicyclohexylcarbodiimide/kg body weight in ethanol, 5 days per week for 13 weeks. All 24 mg/kg male and female mice died or were found moribund and sacrificed prior to day 16. Final mean body weights of 6 and 12 mg/kg males and mean body weight gains of 6 and 12 mg/kg males and females were significantly less than those of the vehicle controls. The predominant clinical pathology changes suggest a secondary, treatment-related inflammatory leukogram and minimal decreased erythron of chronic inflammation that would be consistent with necrosis and chronic active inflammation of the skin. Dermal administration of dicyclohexylcarbodiimide significantly decreased the weight of the epididymis in 6 and 12 mg/kg males and significantly decreased epididymal spermatozoal motility in 6 mg/kg males. Significantly increased incidences of skin lesions at the site of application included epidermal hyperplasia in all dosed groups except those administered 24 mg/kg, chronic active inflammation in all dosed groups except 1.5 mg/kg females, and epidermal necrosis in 24 mg/kg males and females. 20-WEEK STUDY IN FEMALE TG.AC HEMIZYGOUS MICE Groups of 10 female Tg.AC hemizygous mice were dermally administered 0, 0.75, 1.5, 3, 6, or 12 mg dicyclohexylcarbodiimide/kg body weight in ethanol, 5 days per week for up to 20 weeks. Due to the severity of skin lesions observed in 12 mg/kg animals, the application of dicyclohexylcarbodiimide was discontinued after eight dermal applications in this group. There were no deaths considered related to dicyclohexylcarbodiimide administration, although 13 animals died or were sacrificed moribund prior to the end of the study: three each from the vehicle control and 0.75 mg/kg groups, four from the 3 mg/kg group, two from the 6 mg/kg group, and one from the 12 mg/kg group. Overall, the survival was within the range known for the Tg.AC hemizygous mouse. Mean body weights of dosed groups of mice were similar to those of the vehicle controls. At the site of application, the incidences of squamous cell papilloma were increased in a dose-related manner. The incidences of chronic active inflammation of the dermis and epidermal hyperplasia were significantly increased in mice administered 3 or 6 mg/kg. 27-WEEK STUDY IN FEMALE p53 HAPLOINSUFFICIENT MICE Groups of 15 female mice were dermally administered 0, 0.75, 1.5, 3, 6, or 12 mg dicyclohexylcarbodiimide/kg body weight in ethanol, 5 days per week for up to 27 weeks. Dosing of the 6 and 12 mg/kg groups was discontinued after 11 and 8 days, respectively, because of the severity of skin lesions at the site of application. Twelve animals died or were sacrificed moribund prior to the end of the study: three from the 3 mg/kg group, one from the 6 mg/kg group, and eight from the 12 mg/kg group. Mean body weights of dosed groups of mice were similar to those of the vehicle controls. No neoplasms were attributed to administration of dicyclohexylcarbodiimide. At the site of application, the incidences of focal epidermal hyperplasia were significantly increased in 1.5, 3, and 12 mg/kg mice, the incidences of focal chronic active inflammation of the dermis were increased in groups administered 3 or 12 mg/kg, and the incidences of focal ulcer and focal chronic active inflammation of the subcutaneous tissue were increased in the 12 mg/kg group. GENETIC TOXICOLOGY Dicyclohexylcarbodiimide was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535, with or without rat or hamster liver S9 activation enzymes. In vivo, there was a small but significant increase in the frequency of micronucleated normochromatic erythrocytes in male and female B6C3F 1 mice after 13 weeks of dermal exposure to dicyclohexylcarbodiimide. Negative results were obtained, however, in an acute three-injection micronucleus study in bone marrow of male F344/N rats. CONCLUSIONS Under the conditions of this 27-week dermal study, there was no evidence of carcinogenic activity* of dicyclohexylcarbodiimide in female p53 haploinsufficient mice administered 0.75, 1.5, 3, 6, or 12 mg/kg in ethanol. Female Tg.AC hemizygous mice dermally dosed with dicyclohexylcarbodiimide for 20 weeks had significantly increased incidences of squamous cell papilloma of the skin at the site of application. Nonneoplastic lesions noted at the site of application included chronic active inflammation and epidermal hyperplasia in female p53 haploinsufficient mice and female Tg.AC hemizygous mice.

Acute stress modulates the irritant component of sensitizers in allergic contact dermatitis: implications for exposure assessment.

Exposure of skin to noxious environmental stimuli can cause allergic contact dermatitis (ACD), which is a major health risk. Epidemiological studies have determined that 40% of workers report that their jobs are very, or extremely, stressful, and the number of chemicals to which workers are exposed increases each year. We hypothesized that combined exposure to a workplace stressor and a sensitizing chemical would alter the time course and magnitude of the skin immune response. We assessed the mixed exposure of chemical and restraint stress using three potent skin sensitizers, 2,4 dinitrofluorbenzene (DNFB), dicyclohexylcarbodiimide (DCC), and oxazolone, (OXA) on the ear swelling response in stress-susceptible BALB/c mice. Quantitative analyses showed that the dose-response relationship for each chemical followed a cubic trend. Although stress did not alter the shape of the curve, application of restraint stress on day 1 or on day 6 diminished the ear swelling response to 0.1% DNFB. However, if the concentration of the challenge dose was increased to a more irritating concentration, 0.25% DNFB, ear swelling was enhanced. Restraint stress applied on day 6 also increased ear swelling in response to the highly irritating sensitizer DCC, but not to the low-irritancy chemical OXA. These data support the hypothesis that dose-response relationships exist for sensitization with chemical and that restraint stress modulation of the ear swelling response is both chemical specific and dependent on the irritancy potential of the chemical.

MHC restriction in contact hypersensitivity to dicyclohexylcarbodiimide.

Mouse ear swelling tests were performed using different strains of mice with dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), di-p-tolylcarbodiimide (DTC), and positive control chemicals, such as dinitrochlorobenzene (DNCB) and oxazolone (OXA). The chemicals were examined at different doses up to the minimal irritating concentration determined in a irritancy assay. While BALB/c mice exhibited strong responses for the carbodiimide compounds, C3H/HeN mice demonstrated no reactions. Other strains, C57BL/6 and DBA/1, also showed responses to DCC, but CBA/J mice with the same haplotype as C3H/HeN (H-2(k)) did not. Based on our present findings, there may be a specific unresponsiveness to DCC dependent on the H-2(k) haplotype.

Induction of micronucleated erythrocytes in rodents by diisopropylcarbodiimide and dicyclohexylcarbodiimide: dependence on exposure protocol.

The induction of micronucleated erythrocytes by diisopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC) was investigated as part of a U.S. National Toxicology Program (NTP) evaluation of the subchronic toxicity of these chemicals. Analysis of peripheral blood smears from male and female B6C3F1 mice exposed to 17.5-140.0 mg DIC/kg/day by skin painting for 13 weeks revealed dose-related increases in the frequency of micronucleated normochromatic erythrocytes (MN-NCE) in both sexes. Results of a similar 13-week peripheral blood micronucleus (MN) test with DCC (1.5-12.0 mg/kg/day) were also positive, although the increases in MN-NCE were not as great as those observed with DIC. In contrast to the positive results of the subchronic skin-painting studies in mice, acute bone marrow MN studies with DIC and DCC in male F344 rats, using intraperitoneal (i.p.) injection, yielded negative results. Both the acute and the subchronic exposures included doses that produced clinical signs of toxicity. Acute mouse bone marrow MN tests with DIC administered in single or triple i.p. injection protocols were subsequently conducted to determine if the differing responses between mice and rats were due to species or protocol differences. The results of these acute tests were negative or equivocal. Because the subchronic studies produced positive results, it was hypothesized that these carbodiimides required multiple treatments over an extended period of time to produce an increase in MN-erythrocytes. To confirm the original response, a second dermal subchronic study was conducted with DIC; the protocol was modified to include sequential blood samplings to permit monitoring MN frequencies over time. The data demonstrated a small but consistent induction of micronucleated erythrocytes in mice treated with DIC by skin painting.

Contact hypersensitivity to dicyclohexylcarbodiimide and diisopropylcarbodiimide in female B6C3F1 mice.

Dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC) are two commonly used coupling reagents in protein synthesis resulting in exposure of individuals in chemical and pharmaceutical industries as well as research laboratories involved in protein synthesis and recombinant DNA techniques. The objectives of these studies were to determine the irritation and sensitizing potential of these two compounds when applied topically to B6C3F1 mice. Sensitization potential was assessed by the Mouse Ear Swelling Test (MEST) and the murine Local Lymph Node Assay (LLNA). Concentrations used in the contact hypersensitivity assays were determined by primary irritancy studies. DCC and DIC were identified as both irritants and contact sensitizers with the MEST being a more sensitive indicator of sensitization potential. The MEST identified DCC as a sensitizer at concentrations as low as 0.006% (w/v) 24 hr and 48 hr post challenge and DIC at 0.3% (w/v) and 1.5% (w/v) 24 and 48 hr post challenge, respectively. In the LLNA, the lowest concentrations yielding a significant response were 0.06% (w/v) for DCC and 10% (w/v) for DIC.