Induction of chromosome aberrations in cultured human lymphocytes treated with ethoxyquin

The chromosomal aberration test was employed to investigate the effect in vitro of a known antioxidant and food preservative, ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) on human chromosomes. The studies were undertaken because there are no published in vitro data on genotoxicity of EQ in mammalian cells and there are many reports pointing out that it may be harmful to animals and human beings. Lymphocytes obtained from three healthy donors were incubated with EQ (0.01-0.5mM) both with and without metabolic activation. Stability studies performed by HPLC analysis showed that EQ was stable under the conditions of the lymphocyte cultures. The results of the chromosome aberration assay showed that EQ induces chromosome aberrations: gaps and breaks as well as dicentrics and atypical translocation chromosomes.     

Induction of chromosome aberrations in cultured human lymphocytes treated with ethoxyquin

The chromosomal aberration test was employed to investigate the effect in vitro of a known antioxidant and food preservative, ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) on human chromosomes. The studies were undertaken because there are no published in vitro data on genotoxicity of EQ in mammalian cells and there are many reports pointing out that it may be harmful to animals and human beings. Lymphocytes obtained from three healthy donors were incubated with EQ (0.01–0.5 mM) both with and without metabolic activation. Stability studies performed by HPLC analysis showed that EQ was stable under the conditions of the lymphocyte cultures. The results of the chromosome aberration assay showed that EQ induces chromosome aberrations: gaps and breaks as well as dicentrics and atypical translocation chromosomes.     

Genotoxic and antioxidant activities of ethoxyquin salts evaluated by the comet assay

Four newly synthesized salts of ethoxyquin (EQ: 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline), an antioxidant used in animal feeds, were evaluated with the use of the comet assay performed on human lymphocytes: ethoxyquin ascorbate, ethoxyquin hexanoate, ethoxyquin salicylate and ethoxyquin salt of Trolox C (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid). In the study the abilities of these compounds to cause DNA fragmentation and to protect against H2O2-induced DNA damage were analysed. The obtained results were compared with those noted earlier for EQ. After EQ salts treatments (1-25 microM) the genotoxic effects were observed, but the genotoxic potentials of the compounds studied were lower than that of EQ. On the other hand, EQ salts, similarly to EQ, effectively protected the cells from oxidative effect of H2O2. EQ hexanoate was the most effective and its antioxidant activity was even slightly higher than that of EQ. We suggest that it is worth further detailed studies to estimate its usefulness as a preservative.     

5(Z)-benzylidene-1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-1-aza-6-oxa-chrysenes as non-steroidal glucocorticoid receptor modulators

A series of 5-benzylidene-1,2-dihydro-2,2,4-trimethyl-5H-1-aza-6-oxa-chrysenes was synthesized and profiled for their ability to act as selective glucocorticoid receptor modulators (SGRMs). The synthesis and structure-activity relationships for this series of compounds are presented.     

Identification of urinary metabolites of the nephrotoxic hydrocarbon 2,2,4-trimethylpentane in male rats

The compound 2,2,4-trimethylpentane (2,2,4 TMP) is reported to be especially potent in inducing kidney lesions in male rats (1,2). Although the pathology produced by 2,2,4 TMP has been examined (1), there are no reports concerning the metabolism of 2,2,4 TMP by the male rat. The eight principal urinary metabolites of 2,2,4 TMP found in the urine of Fischer 344 male rats are: 2,2,4-trimethyl-1-pentanol, 2,4,4-trimethyl-1-pentanol, 2,4,4-trimethyl-2-pentanol, 2,2,4-trimethyl-1-pentanoic acid, 2,4,4-trimethyl-1-pentanoic acid, 2,4,4-trimethyl-2-hydroxy-1-pentanoic acid, 2,2,4-trimethyl-5-hydroxy-1-pentanoic acid and 2,4,4-trimethyl-5-hydroxy-1-pentanoic acid.     

Comparative analysis of cytotoxic, genotoxic and antioxidant effects of 2,2,4,7-tetramethyl-1,2,3,4-tetrahydroquinoline and ethoxyquin on human lymphocytes

2,2,4,7-Tetramethyl-1,2,3,4-tetrahydroquinoline (THQ) is a new synthetic compound with potential antioxidant activity. In this study, cytotoxic, genotoxic and antioxidant activities of THQ were studied on human lymphocytes with the use of the trypan blue exclusion assay, the TUNEL method, the comet assay and the micronucleus test. The activities of THQ were compared with those of a structurally similar compound-ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ), which is used in animal feeds as a preservative. Cytotoxic effects of THQ were observed after 1-h treatment at the concentration of 500 microM and after 24-h treatments at the concentrations of 250-500 microM. Although the micronucleus test did not reveal a genotoxic effect of THQ, in the comet assay the statistically significant increase in DNA damage was observed as compared with the control. On the other hand, the protection of human lymphocytes against DNA damage induced by hydrogen peroxide suggests an antioxidant activity of THQ. The comparative analysis of THQ and EQ activities performed in these studies revealed that THQ was less cytotoxic and less genotoxic than EQ. Slightly lower antioxidant activity of THQ was also shown in the comet assay when it was used at the lower studied doses (1-5 microM), but for the highest one (10 microM) its efficiency was similar to that of EQ. In the micronucleus assay THQ was more effective than EQ in protecting the cultured lymphocytes from clastogenicity of H2O2. We believe that THQ is worthy of further detailed studies on its antioxidant properties to confirm its usefulness as a preservative.     

Synthesis and antitrypanosomal evaluation of derivatives of N-benzyl-1,2-dihydroquinolin-6-ols: Effect of core substitutions and salt formation

Analogs of the trypanocidal lead compound 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate were prepared to extend the structure-activity relationship in this series of molecules, improve the in vivo antitrypanosomal activity of the lead, and determine whether ester prodrugs are needed to overcome the instability of the dihydroquinolin-6-ols. Two of the most active compounds identified in this study were 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-(2-methoxy)benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride. These stable solids possessed low nanomolar IC₅₀ values against Trypanosoma brucei rhodesiense STIB900 in vitro and provided cures in an early treatment acute mouse model of African trypanosomiasis when given ip at 50 mg/kg/day for four consecutive days.     

Champanones, yellow pigments from the seeds of champa (Campomanesia lineatifolia)

Chemical investigation of the methanol extract of the seeds of Campomanesia lineatifolia Ruiz and Pav. (Myrtaceae) led to the isolation of two new beta-triketone type compounds, named champanones A (1) and B (2), together with the known 2,3-dihydro-5-hydroxy-6,8,8-trimethyl-2-phenyl-4H-1-benzopyran-4,7(8H)-dione (champanone C) (3). The structures of 1 and 2 were determined to be 2,2,4,4-tetramethyl-6-(1-oxo-3-phenylprop-2-enyl) cyclohexane-1,3,5-trione (occurs as an enol form) and 2,2,4-trimethyl-6-(1-oxo-3-phenylprop-2-enyl)cyclohexane-1,3,5-trione (occurs as an enol form), respectively, by means of spectroscopic analysis. The three compounds showed mild antimicrobial activity.     

Effect of dietary vitamin E and Santoquin on regenerating rat liver

The influence of dietary vitamin E and Santoquin on lipid peroxidation and liver regeneration in partially-hepatectomized rats was studied. Rats were fed either a basal 10% tocopherol-stripped corn oil diet, the basal diet plus 40 mg dl-alpha-tocopheryl acetate/kg, or the basal diet plus 2 g Santoquin (6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline)/kg. After 6 weeks, rats fed the antioxidant-deficient diet produced more of the lipid peroxidation product, pentane, than did the rats fed antioxidants. Partial hepatectomy was performed after six and one-half weeks or ten weeks of feeding the diets. At 3 and 6 days after surgery, pentane production was significantly elevated over pre-surgery levels in rats fed the antioxidant-deficient or vitamin E-supplemented diets, but not in rats fed the Santoquin-supplemented diet. Six days after surgery, there were fewer thiobarbituric acid reactants in regenerating liver of Santoquin-fed rats than of vitamin-E fed rats or antioxidant-deficient rats. There was no increase in the 6-day level of thiobarbituric acid reactants over the 3-day level in livers of rats fed Santoquin, while there was an increase in livers of the antioxidant-deficient and vitamin E-supplemented rats. Liver sulfhydryl levels were higher at 3 and 6 days post surgery in the Santoquin-fed rats than in the antioxidant-deficient or vitamin E-supplemented rats. Plasma gamma-glutamyl-transpeptidase activity was not different among the groups of rats. Between the third and sixth day following surgery, liver regeneration was significantly stimulated in Santoquin-fed, but not vitamin E-fed rats. After 11 days, a stimulatory, but not statistically significant, effect of vitamin E was found. Although DNA content of liver was higher at 6 days than at 3 days post surgery, it was not different among the dietary groups, indicating that cell proliferation rather than hypertrophy had occurred. Partial hepatectomy could have altered the ability of the liver to metabolize pentane, thus explaining part of the increased production of pentane. However, the results obtained support the interpretation that elevated levels of dietary antioxidants can be beneficial in terms of reduced lipid peroxidation and increased rates of liver regeneration following liver surgery.     

The metabolism of 3-methylcholanthrene and some related compounds by rat-liver homogenates

1. A chromatographic investigation of the products of the metabolism of 3-methylcholanthrene by rat-liver homogenates showed the formation of compounds with the properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene and 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene. A glutathione conjugate that is probably S-(11,12-dihydro-12-hydroxy-3-methyl-11-cholanthrenyl)glutathione was also detected. 3-Methylcholanthrene-1- and -2-one and -1,2-quinone were also present, but these products may have arisen by the chemical oxidation of the corresponding hydroxy compounds. 2. Other metabolic products were tentatively identified as 9- and 10-hydroxy-3-methylcholanthrene, 4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene and 3-hydroxymethylcholanthrene. 3. 1- and 2-Hydroxy-3-methylcholanthrene were converted by homogenates into the related ketones and into products with the properties of cis- and trans-1,2-dihydroxy-3-methylcholanthrene: 3-methylcholanthren-1- and -2-one were converted into their related hydroxy compounds and into the isomeric 1,2-dihydroxy compounds. The isomeric 1,2-dihydroxy compounds were each partly converted into the other isomer by these homogenates. All the above substrates also yielded products that appeared to be derivatives of 3-hydroxymethylcholanthrene. 4. 3-Methylcholanthrylene was converted by rat-liver homogenates into products with the properties of trans-1,2-dihydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene and 3-methylcholanthren-2-one. A small amount of the cis-1,2-dihydroxy compound was also formed, together with a glutathione conjugate that is possibly S-(2-hydroxy-3-methyl-1-cholanthrenyl)glutathione or its positional isomer. 5. An unidentified product was detected in the metabolism of 3-methylcholanthrene, the monohydroxy compounds, the ketones and the dihydroxy compounds, the formation of which appeared to involve metabolism at the 1,2-bond. 6. 11,12-Epoxy-11,12-dihydro-3-methylcholanthrene was converted by rat-liver homogenates into products with the properties of 11-hydroxy-3-methylcholanthrene (or, less likely, the 12-isomer), 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene and the glutathione conjugate described above. Products with the properties of these compounds were formed when the epoxide was allowed to react with glutathione in an aqueous medium. 7. Mouse-liver homogenate converted 3-methylcholanthrene into products with the chromatographic properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene, 3-methylcholanthrene-1- and -2-one and -1,2-quinone and the unidentified hydroxy-3-methylcholanthrenes. 8. The syntheses of cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 3-methylcholanthren-2-one, 2-hydroxy-3-methylcholanthrene, 3-methylcholanthrylene, 11,12-epoxy-11,12-dihydro-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene are described.     

Chemotactic selection of Tetrahymena pyriformis GL induced with histamine, di-iodotyrosine or insulin

Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline (EQ) is a synthetic antioxidant used for preventing rancidity in animal foodstuffs. Three groups of ten fish were given a diet containing respectively 75 (control group with the commercial food), 200 and 400 ppm EQ for 16 days. The control group had a plasma osmolality and chloride concentration within the normal range of marine teleosts, but sodium concentrations of only about 110 mM, indicating the presence in the plasma of substantial amounts of another cation. Fish given food with 400 ppm EQ displayed a 70 mM increase in the plasma concentration of sodium. This indicates that EQ has disturbed the iono-regulatory mechanisms, probably by reducing the ATP production or inhibiting directly the Na/K-ATPase in the gills. The large increase in plasma sodium concentration was not accompanied by any significant increase in plasma osmolality, indicating that at least a part of the sodium added to the plasma is made osmotically inactive. In spite of the elevated plasma sodium concentration, the sodium content of erythrocytes of the 400-ppm EQ fish was reduced to half, while the content of calcium was unaffected. The transmembrane energy gradient of sodium in the EQ exposed turbot obviously increased, allowing them to use a sodium coupled antiport system to keep the cellular calcium content low when the Ca-ATPases is blocked. A mechanism of this kind is also likely to be important to turbot that experience hypoxia under natural conditions. The 400-ppm group also displayed a substantial increase in liver weight, but the physiological significance of this effect is not clear. The leucocyte counts indicated the absence of obvious immunological effects.     

Three isoflavanones with cannabinoid-like moieties from Desmodium canum

Three further derivatives of 5,7,2',4'-tetrahydroxy-6-methyl isoflavanone have been isolated from the root extract of Desmodium canum and assigned the structures 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(1a,2,3,3a,8b,8c-hexahydro-6-hydroxy-1,1,3a-trimethyl-1H-4-oxabenzo[f]cyclobut[c,d]inden-7-yl)-4H-1-benzopyran-4-one (1) 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(6a,7,8,10a-tetrahydro-3-hydroxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-2-yl)-4H-1-benzopyran-4-one (2) 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(3-hydroxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-2-yl) 4H-1-benzopyran-4-one (3). The three compounds and the previously isolated chromene 4 all derive from the geranylated precursor 5 by a series of cannabinoid-like oxidative rearrangements.     

Dioxygenative cleavage of C-methylated hydroquinones and 2,6-dichlorohydroquinone by Pseudomonas sp. HH35.

The dioxygenolytic catabolism of five C-methylated hydroquinones and 2,6-dichlorohydroquinone in Pseudomonas sp. strain HH35 was elucidated. This organism, which is known to catabolise 2,6-dimethylhydroquinone by 1,2-cleavage, accumulated metabolites from 2-methyl-, 2,3-dimethyl-, 2,5-dimethyl-, 2,3,5-trimethyl- and 2,3,5,6-tetramethylhydroquinone which we isolated and characterised by mass spectrometry and (1)H NMR and UV spectroscopy. The identification of these metabolites defined the impact of methyl groups present in the hydroquinone and showed how each substitution pattern determined the site of the initial enzymic attack. With the exception of the 2,3,5,6-tetramethylhydroquinone, all C-methylated hydroquinones were catabolised by an initial dioxygenolytic cleavage occurring adjacent (1,2- or 3,4-cleavage) to a hydroxy group. In addition, our results indicated that the 2,6-dichlorohydroquinone is catabolised in a similar way by this strain.     

Studies on the Oxidation Mechanism of Vitamin E

α-Tocopherol model compound, 2,2,5,7,8-pentamethyl-6-hydroxychroman was oxidized under oxygen bubbling. Four oxidation products of 2-(γ,γ-dimethylallyl)-3,5,6-trimethyl-1,4-benzoquinone (B), 2,2,7,8-tetramethyl-5-formyl-6-hydroxychroman (C), trimer (D) and tocopherylethane (G) were identified, and spirodimer (E) was tentatively identified by TLC. Two of them, (B) and (C) have not been obtained in the oxidation of α-tocopherol model compound with p-quinone, alkaline ferricyanide and other compounds as oxidizing agent. A scheme of oxidation mechanism of α-tocopherol model compound was also proposed.     

Analysis of insecticidal Azadirachta indica A. Juss. fractions

As a result of chemical investigation on the ethanolic extract of fresh fruit coatings of Azadirachta indica A. Juss. (neem), twenty-seven compounds were identified in non-polar to less polar fractions which showed pesticidal activity determined by WHO method against Anopheles stephensi Liston. These identifications were basically made through GC-EIMS and were further supported by other spectroscopic techniques, including 13C NMR, UV and FTIR as well as retention indices. Thus sixteen n-alkanes, 1-16; three aromatics 2,6-bis-(1,1-dimethylethyl)-4-methyl phenol (17), 2-(phenylmethylene)-octanal (20), 1,2,4-trimethoxy-5-(1Z-propenyl)-benzene (27); three benzopyranoids 3,4-dihydro-4,4,5,8-tetramethylcoumarin (18), 3,4-dihydro-4,4,7,8-tetramethylcoumarin-6-ol (19), 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta[g]-2-benzopyran (22); one sesquiterpene methyl-3,7,11-trimethyl-2E,6E,10-dodecatrienoate (21); three esters of fatty acids methyl 14-methyl-pentadecanoate (23), ethyl hexadecanoate (24), ethyl 9Z-octadecenoate (25) and one monoterpene 3,7-dimethyl-1-octen-7-ol (26) were identified. Except 6, 8, 24 and 25 all these compounds were identified for the first time from the pericarp and fifteen of these, 1-3, 7, 9, 10, 17-23, 26, 27, are hitherto unreported previously from any part of the tree. Although this tree is a rich source of various natural products, it is the first report of identification of mono- and sesquiterpenes 26 and 21 and a potent antioxidant, 17.     

Mutagenicity of K-region derivatives of 1-nitropyrene; remarkable activity of 1- and 3-nitro-5H-phenanthro[4,5-bcd]pyran-5-one

The mutagenic activities toward S. typhimurium strains TA98 and TA100 of K-region derivatives of 1-nitropyrene and pyrene were determined. The compounds tested were trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene (Compound 3), trans-4,5-dihydro-4,5-dihydroxypyrene (Compound 4), 1-nitropyrene-4,5-quinone (Compound 5), 1-nitropyrene-9,10-quinone (Compound 6), pyrene-4,5-quinone (Compound 7), and the lactones, 1-nitro-5H-phenanthro[4,5-bcd]pyran-5-one (Compound 8), 3-nitro-5H-phenanthro[4,5-bcd]pyran-5-one (Compound 9), and 5H-phenanthro[4,5-bcd]pyran-5-one (Compound 10). Neither pyrene nor any of its K-region derivatives was mutagenic, either in the absence or presence of S9 mix at the doses tested. Of the K-region derivatives of 1-nitropyrene, the lactones (Compounds 8 and 9) were generally the most active; 0.25 microgram/plate induced 900-2200 revertants in TA98 or TA100 without activation. The 4,5-dihydrodiol (Compound 3), an established mammalian metabolite of 1-nitropyrene, was less mutagenic than was 1-nitropyrene in TA98, but was more mutagenic than was 1-nitropyrene in TA100, regardless of the presence of S9 mix. The quinones (Compounds 5 and 6) were less mutagenic than was 1-nitropyrene in the absence of S9 mix in both strains, but their activities were increased in the presence of S9 mix. The mutagenic activities of the lactones (Compounds 8 and 9) were lower in strains TA98NR and TA98/1,8-DNP6 than in TA98, indicating that nitro-reduction and esterification are involved in their activation. The results of this study indicate that K-region derivatives of 1-nitropyrene may be important in its metabolic activation.     

Aporphinoid alkaloids and other constituents from Lettowianthus stellatus

Two new aporphinoid alkaloids, f1ttowianthine and 11-methoxylettowianthine were isolated from the root bark of Lettowianthus stellatus, together with the new sesquiterpene 11-hydroxyguaia-4,6-diene and the known compounds liriodenine, (Z)-7-octadecen-9-ynoic acid, methyl (2E,6E,10R)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate, methyl (2E,6E,10R)-10,11-dihydroxy-3,7,11-trimethyl-2,6-dodecadienoate , and 3,4,5-trimethoxyphenol. The structure elucidation was achieved by spectroscopic methods.     

Dioxygenative cleavage of C-methylated hydroquinones and 2,6-dichlorohydroquinone by Pseudomonas sp. HH35

The dioxygenolytic catabolism of five C-methylated hydroquinones and 2,6-dichlorohydroquinone in Pseudomonas sp. strain HH35 was elucidated. This organism, which is known to catabolise 2,6-dimethylhydroquinone by 1,2-cleavage, accumulated metabolites from 2-methyl-, 2,3-dimethyl-, 2,5-dimethyl-, 2,3,5-trimethyl- and 2,3,5,6-tetramethylhydroquinone which we isolated and characterised by mass spectrometry and ¹H NMR and UV spectroscopy. The identification of these metabolites defined the impact of methyl groups present in the hydroquinone and showed how each substitution pattern determined the site of the initial enzymic attack. With the exception of the 2,3,5,6-tetramethylhydroquinone, all C-methylated hydroquinones were catabolised by an initial dioxygenolytic cleavage occurring adjacent (1,2- or 3,4-cleavage) to a hydroxy group. In addition, our results indicated that the 2,6-dichlorohydroquinone is catabolised in a similar way by this strain.     

Unusual naphthoquinones, catechin and triterpene from Byrsonima microphylla

The new triterpene Delta1-lupenone (1), together with lupeol, beta-amyrin and betulin were isolated from the wood of Byrsonima microphylla (Malpighiaceae). The new compounds 3-hydroxy-2-methoxy-8,8,10-trimethyl-8H-antracen-1,4,5-trione (2), 3,7-dihydroxy-2-methoxy-8,8,10-trimethyl-7,8-dihydro-6H-antracen-1,4,5-trione (3), (2S*,10aR*)-2,8-dihydroxy-6-methoxy-1,1,7-trimethyl-2,3,10, 10a-tetrahydro-1H-fenantren-9-one (4) and (2S,3S)-3'-hydroxy-4',5,7-trimethoxy-flavan-3-ol (5) were also isolated through monitored TLC using the antioxidant beta-carotene reagent. The antioxidant potential of the compounds 2-5 was measured and none of them showed activity. The structures of these compounds were elucidated by chemical and spectroscopic analysis based on NMR techniques (1H, 13C NMR, COSY, nOe difference, HMQC and HMBC), UV and MS.