Novel aminoarylcysteine adducts in globin of rats dosed with naphthylamine and nitronaphthalene isomers.

Novel aminonaphthylcysteine (ANC) adducts, formed via naphthylnitrenium ions and/or their metabolic precursors in the biotransformation of naphthylamines (NA) and nitronaphthalenes (NN), were identified and quantified in globin of rats dosed intraperitoneally with 0.16 mmol/kg b.w. of 1-NA, 1-NN, 2-NA and 2-NN. Using HPLC-ESI-MS2 analysis of the globin hydrolysates, S-(1-amino-2-naphthyl)cysteine (1A2NC) together with S-(4-amino-1-naphthyl)cysteine (4A1NC) were found in rats given 1-NA or 1-NN, and S-(2-amino-1-naphthyl)cysteine (2A1NC) in those given 2-NA or 2-NN. The highest level of ANC was produced by the most mutagenic and carcinogenic isomer 2-NA (35.8 ± 5.4 nmol/g globin). The ratio of ANC adduct levels for 1-NA, 1-NN, 2-NA and 2-NN was 1:2:100:3, respectively. Notably, the ratio of 1A2NC:4A1NC in globin of rats dosed with 1-NA and 1-NN differed significantly (2:98 versus 16:84 respectively), indicating differences in mechanism of the adduct formation. Moreover, aminonaphthylmercapturic acids, formed via conjugation of naphthylnitrenium ions and/or their metabolic precursors with glutathione, were identified in the rat urine. Their amounts excreted after dosing rats with 1-NA, 1-NN, 2-NA and 2-NN were in the ratio 1:100:40:2, respectively. For all four compounds tested, haemoglobin binding index for ANC was several-fold higher than that for the sulphinamide adducts, generated via nitrosoarene metabolites. Due to involvement of electrophilic intermediates in their formation, ANC adducts in globin may become toxicologically more relevant biomarkers of cumulative exposure to carcinogenic or non-carcinogenic arylamines and nitroarenes than the currently used sulphinamide adducts.

Label-Free Detection of Protein Tyrosine Phosphatase 1B (PTP1B) by Using a Rationally Designed Förster Resonance Energy Transfer (FRET) Probe.

A highly selective detection method of native protein tyrosine phosphatase 1B (PTP1B) is described using a target specific probe equipped with 1-naphthylamine (λex =330 nm, λem =445 nm). Irradiation of a mixture of PTP1B and Probe 1 with ultraviolet light of 280 nm (corresponding to PTP1B excitation maximum) resulted in significant fluorescence increase at 445 nm, following FRET characteristics. This phenomenon does not occur with other closely related phosphatases or cellular abundant alkaline phosphatase (APP). Probe 1, the most potent and selective probe, was found to competitively inhibit PTP1B (Ki ≈42 nm), whereas APP inhibition was found to be in the low micromolar range. Furthermore, Probe 1 discriminates between PTP1B and several other phosphatases. Here, we report real-time label-free FRET detection of pure PTP1B as well as induced human PTP1B in Escherichia coli cell lysate. In contrast to 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a representative fluorescence turn-on PTP substrate, our FRET probe successfully differentiated human cervical carcinoma cell lysate, SiHa, which has a high expression level of PTP1B, from PTP1B-knockdown SiHa cell lysate (that is, siRNA was used for PTP1B knockdown).

Naphthoquine-induced Central Nervous System and Hepatic Vasculocentric Toxicity in the Beagle Dog.

Naphthoquine phosphate (NP) was considered as a partner drug with a promising antimalarial drug candidate. Here we report unexpected adverse clinical signs and microscopic findings in a canine pilot toxicology study with NP. Male and female dogs were dosed daily by oral gavage with NP at 2, 10, or 50 mg/kg/day for a maximum of 14 days. NP was not tolerated at ≥10 mg/kg/day; several animals were sacrificed in moribund condition and marked neurological clinical signs were noted at 50 mg/kg/day. The main microscopic observation was central nervous system vasculocentric inflammation (mainly lymphocytes and macrophages) in the white and gray matter of various regions of the brain at ≥2 mg/kg/day and at lower incidence in the spinal cord at ≥10 mg/kg/day. Vasculocentric microscopic changes predominantly centered on the centrilobular vein were also observed in the liver at ≥2 mg/kg/day. Females were more sensitive than males with comparable NP plasma exposure. In conclusion, under the conditions of this study, the administration of NP to dogs via daily oral gavage for up to 2 weeks was not tolerated causing moribundity, marked neurological clinical signs, and vasculocentric microscopic changes in the central nervous system and the liver.

Enhanced toxicity to the cyanobacterium Microcystis aeruginosa by low-dosage repeated exposure to the allelochemical N-phenyl-1-naphthylamine.

It has been puzzling whether and how a plant could exert a strong allelopathic inhibition to the target organisms by releasing low concentrations of allelochemicals. Plant allelochemicals have been proposed to be released continuously, however, direct evidence from specific allelochemicals is urgently required. In the present study, the toxicity of allelochemical N-phenyl-1-naphthylamine (NPN) towards the cyanobacterium Microcystis aeruginosa by two different exposure patterns was compared. One was low-dosage repeated exposure (LRE), in which 50  µg L-1 NPN was repeatedly dosed to simulate the continual release of allelochemicals, and the other one was high-dosage single exposure (HSE) as per the routine toxicity assay. The results showed a significant growth inhibition to M. aeruginosa in the LRE group, where the inhibition rate reached above 90% from day 6 to day 9. The cell-membrane damage ratio increased from 64.05% on day 5 up to 96.60% on day 9. PSII photosynthesis activity expressed as Fv/Fm, ΦPSII, NPQ and ETRmax was also thoroughly inhibited in this group. Whereas the growth and PSII photosynthesis activity of M. aeruginosa in the HSE group were inhibited initially, but recovered gradually from day 4 or 5, which was accompanied by a continuous reduction of NPN content in culture solutions. Although NPN content in the LRE group was relatively lower, it remained at a more stable level throughout the experiment. These results indicate that continual release of low-dosage allelochemicals by aquatic plants plays crucial roles in their potent inhibition against cyanobacteria. Low-dosage continual exposure pattern needs to be investigated further.

Comparative biotoxicity of N-Phenyl-1-naphthylamine and N-Phenyl-2-naphthylamine on cyanobacteria Microcystis aeruginosa.

N-Phenyl-1-naphthylamine (P1NA) and N-Phenyl-2-naphthylamine (P2NA) are both widely used as antioxidant and plant secondary metabolites. In this study, growth, esterase, photosynthetic activity and cell membrane integrity were used as biomarkers to compare biotoxicity of P1NA and P2NA on Microcystis aeruginosa. According to the results, a dose-response relationship was observed only between P1NA concentrations and growth inhibition. The EC50 (48 h) of P1NA calculated from growth inhibition was 16.62 µM, while that of P2NA was not detected. When the esterase and photosynthetic activity were applied to evaluate the biotoxicity, it was found that a concentration of 20 µM P1NA, P2NA caused reduction of esterase activity and Fv/Fm of M. aeruginosa to 22.2 and 3.3%, 97.5 and 92.1%, respectively, after 48 h exposure. The percentage of membrane-damaged cells was increased as P1NA exposure concentration increased, but that was not detected when exposure to P2NA. The difference substituted position in the molecular structure of P1NA and P2NA leads to different toxicological properties and only P1NA was found highly toxic to M. aeruginosa. The toxicity is due to that only P1NA can be biotransformed to 1,4-naphthoquinone, which could induce overproduction of intracellular ROS as well as result in oxidative damage and growth inhibition of test organism.

Pulmonary adenoma response of strain A mice to sulfonic acid derivatives of 1- and 2-naphthylamines.

Various sulfonic acid derivatives of 1-naphthylamine and 2-naphthylamine were tested in inbred A/St (male and female) mice by the pulmonary adenoma bioassay to determine if this class of compounds, used as intermediates in the dye-stuff industry, possesses tumorigenic activity. Neither 1-naphthylamine nor the four sulfonic acid derivatives of 1-naphthylamine tested were tumorigenic. However, 2-naphthylamine and two of the three sulfonic acid derivatives of 2-naphthylamine tested produced statistically significant lung tumor responses at comparable doses. These results indicated that this class of compounds should be examined more extensively for carcinogenic activity.

Failure of pure 1-naphthylamine to induce bladder tumors in dogs.

1-Naphthylamine was administered p.o. to beagles for approximately 9 years at a daily dose of 15 mg/kg (5 days a week). At autopsy, no tumors or other pathological changes were observed in the bladders of any of these animals. With the possible exception of the excessive accumulation of lipofuscin in the hepatocytes of these dogs, no test compound-related pathological changes in other tissues of the body were observed. Analysis of the urine revealed the presence of small amounts of N-oxidation products (1-nitrosonaphthalene plus N-hydroxy-1-naphthylamine), ranging from 2.85 to 125 microgram, following a dose of 1-naphthylamine. The theoretical significance of these findings is discussed.

Carcinogenicity of N-phenyl-1-naphthylamine and N-phenyl-2-naphthylamine in mice.

Technical N-phenyl-1-naphthylamine ( PANA ), which is an optic isomer of N-phenyl-2-naphthylamine ( PBNA ), has been used as a rubber additive without suspicion of its being carcinogenic. When male ICR mice were given repeated s.c. injections of both technical and pure PANA in dimethyl sulfoxide, it resulted in high percentage of malignant tumors similar to that in mice given technical PBNA . PANA had a tendency to induce hemangiosarcoma. Similar injections of PANA and PBNA into male TA-1 mice gave similar results. Previous unilateral nephrectomy enhanced both PANA and PBNA induction of renal hemangiosarcomas. The similar carcinogenic potency of PANA and PBNA suggests other routes of metabolic activation besides dephenylation for both chemicals in mice.

Local carcinogenicity, rates of absorption, extent and persistence of macromolecular binding, and acute histopathological effects of N-hydroxy-1-naphthylamine and N-hydroxy-2-naphthylamine.

The N-hydroxy derivatives of 1- and 2-naphthylamine (NA) are directly carcinogenic at sites of application. In this study, the carcinogenicity of these two compounds at s.c. injection sites was compared with their relative rates of absorption, with the extent and persistence of their binding to protein, RNA, and DNA in the skin-subcutis, and with acute histopathological changes observed after local application. Male Sprague-Dawley rats were given injections of the N-hydroxy derivatives in the right rear leg at 16 mumol/dose. When administered twice weekly for 12 weeks, N-hydroxy-1-NA caused a 100% incidence (30 of 30) of poorly differentiated sarcomas at the injection site. N-Hydroxy-2-NA administered in a similar manner resulted in a low yield of tumors (7%; 2 of 30). Injection of N-hydroxy-1-NA once weekly for 12 weeks or twice weekly for 6 weeks also induced a high incidence of sarcomas (93 to 97%), but the time to tumor formation was significantly longer (p less than 0.0001) than in animals treated twice weekly for 12 weeks. The tumors were classified as malignant fibrous histiocytomas. Possible antagonistic or synergistic effects between the two compounds were also investigated. A sequential 6-week treatment with each of the N-hydroxy derivatives did not alter the expected tumor yields. However, alternating injections over 12 weeks caused a significant lengthening in the time to tumor formation (p less than 0.05). N-Hydroxy-1-NA bound covalently to protein, RNA, and DNA to a much greater extent than did N-hydroxy-2-NA. Protein binding with both derivatives decreased by 80 to 90% by 7 days after treatment. RNA binding in N-hydroxy-1-NA-treated rats markedly decreased, while N-hydroxy-2-NA-bound residues diminished only slightly. During this period, the extent of DNA binding with both derivatives remained fairly constant. When N-hydroxy-2-NA was injected 3 days after N-hydroxy-1-NA, there was a marked reduction in the apparent levels of N-hydroxy-1-NA bound to RNA and DNA. The greater tumorigenicity of N-hydroxy-1-NA versus N-hydroxy-2-NA correlated with its greater extent of macromolecular binding. Examination of acute histopathological changes occurring after single injections of N-hydroxy-1-NA and/or N-hydroxy-2-NA indicated that both compounds caused extensive necrosis in tissues at the injection site.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of retrovirus-induced syncytium formation by photoproducts of a brominated 1,8-naphthalimide compound.

A major disadvantage of conventional phototherapy is the requirement for the in situ delivery of stimulating photoenergy subsequent to the binding of photochemicals to target malignant cells, or virus-infected cells, or viruses. This drawback has resulted in considerable limitation in the use of photochemicals in photomedicine. To circumvent this problem, we have investigated the antiviral efficacy of a brominated 1,8-naphthalimide photocompound, termed LY66Br [3-bromo-4-(hexylamino)-N-hexyl-1,8-naphthalimide], which upon exposure to visible light at 420 nm generates independently of oxygen one or more stable antiviral molecular photoproducts (e.g., is 'preactivated'). Human cell lines infected with the human immunodeficiency virus type 1 (HIV-1), or with the human T-lymphotropic virus type-1 (HTLV-I) exposed to photochemical products of LY66Br (P-LY66Br) completely lost their ability to form syncytia in vitro. Photoproducts of P-LY66Br retain full antiviral activity for at least 3 and 6 weeks when stored at room temperature and at -80 degrees C, respectively. Concentrations of P-LY66Br, effective in inhibiting syncytium formation mediated by HIV-1 and HTLV-I, were nontoxic to normal red cell components of whole blood (red blood cell 2,3-diphosphoglyceric acid, adenosine triphosphate, osmotic fragility or blood type antigens). Additionally, no evidence of acute toxicity was demonstrated in mice following an intravenous bolus inoculation to achieve plasma concentration of 600 microM of P-LY66Br. These findings represent the first demonstration of inhibition of retrovirus-induced syncytium formation by a photochemical product, and justify further investigation of the preactivation process of photochemicals in the treatment of systemic viral infections such as the acquired immunodeficiency syndrome (AIDS), in cancer therapy, and in sterilization of banked blood products.

A host-mediated in vivo/in vitro assay with peritoneal murine macrophages for the detection of carcinogenic chemicals.

We have developed a host-mediated assay system for the detection of the transforming action of chemical carcinogens on peritoneal macrophages. Directly as well as indirectly acting carcinogenic substances administered intraperitoneally to NMRI mice could be examined in this way. Resident macrophages were recovered by peritoneal lavage from treated and untreated mice and were cultured in soft agar. After 5-6 days normal and transformed cells could be distinguished. Statistical analysis comparing cells, for example, from alpha-naphthylamine or diphenylhydantoin-treated animals with those from control mice proved that the test is positive at least on a significance level of 5% using the t-test. Further substances revealing a cell-transformation potential were benzene, benz(a)pyrene, 2,3,7,8-tetrachlorodibenzodioxin, N-nitrosodimethylamine, ethidium bromide, aflatoxin B1,N-methyl-N-nitrosourea, 1-methyl-3-nitro-1-nitrosoguanidine, 2-naphthylamine, dieldrin, suramin and trichloroethylene. A weak transforming potential was found for chlorambucil as well as for tetrachloroethylene. With toluene or azidothymidine no cell transformation could be observed. Several immortal cell lines could be established form NMRI mice treated with alpha-naphthylamine or N-methyl-N-nitrosourea. Athymic nu/nu mice injected subcutaneously with these cells developed tumors, establishing the oncogenic potential of these cell lines.

Diaminonaphthalenes and related aminocompounds: mutagenicity, CYP1A induction and interaction with the Ah receptor.

Using 1- and 2-aminonaphthalene as model substrates, we investigated the effect of insertion of a second amino group on mutagenicity, binding to the cytosolic Ah receptor and CYP1A inducibility, and the effects were compared to those elicited by 3,3'-diaminobenzidine and 1-naphthylethylenediamine. 1,5- and 1,8-diaminonaphthalene were effective inducers of CYP1A activity, more potent than 1-aminonaphthalene. 2,3-Diaminonaphthalene was also an inducer of CYP1A, but the effect was similar to that elicited by 2-aminonaphthalene. In contrast, 3,3'-diaminobenzidine and 1-naphthylethylenediamine did not induce CYP1A activity. All aminonaphthalenes displaced [3H]TCDD from the Ah receptor, whereas 3,3'-diaminobenzidine and 1-naphthylethylenediamine failed to do so. The latter two compounds did not elicit a mutagenic response in the Ames test. Introduction of a second amino group at the 3-position of 2-aminonaphthalene did not modulate its mutagenicity. In the case of the non-mutagenic 1-aminonaphthalene, introduction of a second amino group at position 5 had no effect but when it was incorporated at position 8, mutagenic potential was conferred to the molecule. Computer modelling of the putative active site of CYP1A2 revealed that 1,5-diaminonaphthalene is orientated so that the distance of the second amino group from the iron-oxene is 4.037 A while in the case of 1,8-diaminonaphthalene the distance is shorter, 2.744 A, favouring its activation through N-hydroxylation. Of the compounds studied, 1,8-diaminonaphthalene and, to a lesser extent, 2,3-diaminonaphthalene autoinduced their activation. It is concluded that insertion of a second amino group at the 5- or 8-position of 1-aminonaphthalene may enhance biological activity but in the case of 2-aminonaphthalene insertion of a second amino group at position 3 had no major effect.

Local anesthetic activity and acute toxicity of N-substituted 1,2,3,4-tetrahydro-1- and 2-naphthylamines.

Seven N-substituted 1,2,3,4-tetrahydro-1- and three 2-naphthylamines were prepared and tested for local anesthetic activity in the rabbit corneal reflex test and the mouse sciatic nerve block test. At 0.1 and 1%, three 1-alkylamino compounds had durations of action comparable to that of tetracaine in the rabbit corneal reflex test and were considerably more potent than lidocaine. The other four 1-alkylamino derivatives were inactive or at best minimally active. The durations of action of 1% concentrations of the three 2-alkylamino compounds were equivalent to that of 1% lidocaine in the corneal reflex test. In the mouse sciatic nerve block test, the three active 1-alkylamino compounds were considerably longer acting than either tetracaine or lidocaine. Three 1-alkylamino and the three 2-alkylamino compounds showed toxicity equal to or greater than lidocaine, while two 1-alkylamino and two 2-alkylamino compounds showed toxicity equal to or greater than tetracaine by the intraperitoneal route in mice. N-Heptyl-1,2,3,4-tetrahydro-6-methoxy-1-naphthylamine methanesulfonate was the most promising local anesthetic in these series.

Comparison of the sensitivity of Salmonella typhimurium strains YG1024 and YG1012 for detecting the mutagenicity of aromatic amines and nitroarenes.

Salmonella typhimurium YG1024 is a derivative of S. typhimurium TA98 with a high level of N-hydroxyarylamine O-acetyltransferase (OAT) activity. We have demonstrated that this strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes. In this paper, we compared the sensitivities of YG1024 with those of S. typhimurium YG1012, which has about 4 times higher OAT activity than YG1024 but lacks plasmid pKM101. It turned out that YG1024 was more sensitive to the mutagenic actions of 1-aminonaphthalene, 1-nitropyrene, 1,8-dinitropyrene and 2-nitronaphthalene than YG1012 and showed comparable sensitivity to 2-hydroxy-acetylaminofluorene, 2-aminoanthracene and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) to YG1012. These results suggested that YG1024 is more suitable than YG1012 for the efficient detection of mutagenic aromatic amines and nitroarenes.

Transformation of arylamines into direct-acting mutagens by reaction with nitrite.

Arylamines including aniline (I), 1-naphthylamine (II), 2-naphthylamine (III), 2-aminofluorene (IV), 1-aminoanthracene (V) and 1-aminopyrene (VI) were treated with 4 equivalent amounts of nitrite at pH3 and 37 degrees C for 4 h. The reaction mixtures of I, IV, V and VI showed mutagenicity to Salmonella typhimurium TA98 and TA100 strains without metabolic activation. The numbers of His+ revertant colonies to TA98 strain were 110/0.05 mumole I, 970/0.055 mumole IV, 620/0.10 mumole V and 870/0.02 mumole VI. These arylamines were converted into mutagens with diazoquinone, diazonium and nitro functions depending on their structures. The mutagen from I was p-diazoquinone (I2). The mutagen from IV was highly unstable fluorene-2-diazonium salt (IV1). The mutagens from V were N3O3-introduced anthracene (V1-1) and 1-nitroanthracene (V2), and those from VI were unidentified nitro-introduced compound (VI1) and 1-nitropyrene (VI2).

Functional and morphological characterization of neuropathy induced with 5-lipoxygenase inhibitor CGS 21595.

Peripheral toxic neuropathy induced in rats with a 5-lipoxygenase inhibitor CGS 21,595 was characterized using special functional tests and pathological procedures. Functional tests included measurement of grip strength, landing foot splay, assessment of sensorimotor and autonomic functions and monitoring of motor activity. Pathological procedures consisted of perfusion fixation, embedding in plastic, teasing of isolated nerve fibers, and light and electron microscopy. Male and female albino rats received the test article orally by gavage on 5 days per week. To characterize the development of the lesion animals treated with 1000 mg/kg were examined and sacrificed at 2-week intervals until termination at 10 weeks. In a separate study, the dose-effect relationship was examined in groups of animals treated with 50,200 or 1000 mg/kg for 10 weeks. Neurotoxicity occurred only in animals treated with 1000 mg/kg and was first detected following 4 weeks of treatment. Although there were no overt clinical signs of neurotoxicity, functional examination detected a reduction of grip strength, increased landing foot splay and reduced motor activity. Neuropathological examination revealed peripheral segmental demyelination affecting predominantly the Schwann cells in the ventral spinal nerve roots. Owing to its unusual localization in the nervous system and to subtlety of functional signs, peripheral segmental demyelination represents a special diagnostic challenge in toxicological safety studies.

Functional reduction of SK3-mediated currents precedes AMPA-receptor-mediated excitotoxicity in dopaminergic neurons.

In primary cultures of mesencephalon small-conductance calcium-activated potassium channels (SK) are expressed in dopaminergic neurons. We characterized SK-mediated currents (I(SK)) in this system and evaluated their role on homeostasis against excitotoxicity. I(SK) amplitude was reduced by the glutamatergic agonist AMPA through a reduction in SK channel number in the membrane. Blockade of I(SK) for 12 h with apamin or NS8593 reduced the number of dopaminergic neurons in a concentration-dependent manner. The effect of apamin was not additive to AMPA toxicity. On the other hand, two I(SK) agonists, 1-EBIO and CyPPA, caused a significant reduction of spontaneous loss of dopaminergic neurons. 1-EBIO reversed the effects of both AMPA and apamin as well. Thus, I(SK) influences survival and differentiation of dopaminergic neurons in vitro, and is part of protective homeostatic responses, participating in a rapidly acting negative feedback loop coupling calcium levels, neuron excitability and cellular defenses. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Lifetime carcinogenicity study of 1- and 2-naphthylamine in dogs.

Groups of male and female beagle dogs were given daily doses of 400 mg of various mixtures of naphthylamines for up to 109 months. Survivors were killed at 128 months. A variety of pathological conditions was diagnosed, but the only effect related to treatment was the induction of bladder neoplasms. All dogs which received pure 2-naphthylamine developed transitional-cell carcinomas of the bladder within 34 months. Two of 8 dogs receiving 6% 2-naphthylamine in 1-naphthylamine developed early carcinoma and 2/8 dogs receiving 0.5% 2-naphthylamine in 1-naphthylamine developed haemangioma of the bladder. Some of the dogs receiving 1-naphthylamine (total dose 950 g) and the controls had focal cystitis or hyperplasia, but no neoplasia of the bladder. These results confirm the carcinogenicity of 2-naphthylamine to dogs. No carcinogenic effect of 1-naphthylamine was observed, indicating that it is at least 200 times less potent as a carcinogen than 2-naphthylamine. The incidence of bladder cancer in dogs fed mixtures of both naphthylamines explains why previous experimental and epidemiological studies of impure 1-naphthylamine have revealed carcinogenicity.