Mechanisms of acute inhalation effects of (+) and (-)-alpha-pinene in BALB/c mice

The effects of (+)-alpha-pinene and (-)-alpha-pinene vapours were studied for respiratory effects in BALB/c mice. The (+) enantiomer showed persistent sensory irritation effect on the upper respiratory tract during exposures in the range of 100 to 3691 ppm. The threshold concentration for this effect was calculated to be about 70 ppm, which is close to the no-effect level of about 40 ppm in humans. A significant airflow limitation occurred from exposure concentrations of 200 ppm and higher. No irritating effect was observed at the alveolar level and no central nervous system effect was obvious. In the exposure range from 218 to 5213 ppm, the (-) enantiomer produced only a short-lasting sensory irritation effect during the first 10 min. of exposure and only at concentrations above 2900 ppm. The tidal volume decreased significantly from approximately 400 ppm, but it was first conspicuous above 1000 ppm. Airflow limitation appeared consistently from approximately 2000 ppm. The (-) enantiomer also induced anesthesia/and or pulmonary irritation as well as sudden death at concentrations above 2600 ppm. Overall, the enantiomers showed different time-dependent and stereoselective effects. The lower sensory irritation effect of the (-) enantiomer probably being due to less of the molecule being adsorbed to a sensory irritant receptor.     

Mutagenicity and cytotoxicity of N-methyl-2-pyrrolidinone and 4-(methylamino)butanoic acid in the Salmonella/microsome assay

The industrial solvent N-methyl-2-pyrrolidinone (NMP) and its hydrolysis product, 4-(methylamino)butanoic acid (N-MeGABA), were examined for mutagenicity and cytotoxicity in the Ames Salmonella/microsome assay. In order to detect a broad range of possible mutagenic endpoints, the following strains were used in the assay: base-pair substitution strains TA100, TA102 and TA104; frameshift strains TA97 and TA98; and repair proficient strains TA2638, UTH8413 and UTH8414. In the standard plate incorporation assay, six log-linear doses of each compound were tested; doses ranged from 0.01 to 1000 mumol/plate for NMP, and 0.01 to 316 mumol/plate for N-MeGABA. Neither compound was detectably mutagenic when tested in the presence and absence of metabolic activation by Aroclor-induced rat liver S9. NMP did show significant responses with strains TA102 and TA104 that were less than two-fold over background, but no clear dose-response relationships were evident. A preincubation modification of the assay was also performed, using strains TA98 and TA104. Mutagenic activity was not observed for NMP, while N-MeGABA showed significant responses with TA104 but dose-related mutagenicity was not established. Preincubation testing revealed both NMP and N-MeGABA to be cytotoxic to the test population of Salmonella at the highest treatment doses.     

Study on the mutagenicity and antimutagenicity of beta-ionone in the Salmonella/microsome assay.

beta-Ionone (BIO) is a degraded (C(13)) sesquiterpenoid compound found in a variety of edible and aromatic plants. BIO and other ionone derivatives have been used in fragrance products and as flavoring food additives. In this study we investigated the mutagenic and antimutagenic activities of BIO using the Salmonella/microsome assay. Mutagenicity was evaluated by two tests with Salmonella typhimurium strains TA100, TA98, TA97a and TA1535, without and with addition of S9 mixture. A first assay was performed by the plate incorporation procedure and a confirmation test by the pre-incubation method. In either test, no increase in the number of his(+) revertant colonies over the negative (solvent) control values was noted with any of the four tester strains thereby indicating that BIO was not genotoxic in the Salmonella assay. Antimutagenic activity was investigated by testing (by the plate incorporation method) different non-toxic doses of BIO against one or more non-toxic doses of direct-acting (sodium azide: SA, 4-nitroquinoline-N-oxide: 4-NQNO, 2-nitrofluorene: 2-NF and nitro-o-phenylenediamine: NPD) as well as indirect-acting (cyclophosphamide: CP, benzo[a]pyrene: B[a]P, aflatoxin B1: AFB1, 2-aminoanthracene: 2-AA, and 2-aminofluorene: 2-AF) mutagens. BIO did not alter the effects of any direct-acting mutagen or B[a]P and 2-AF. Mutagenic effects of AFB1 and CP, however, were markedly and dose-dependently antagonized by BIO. It has been reported that, in the rat liver, activation of B[a]P and 2-AF depend on CYP1A1 activity, and that CYP2B subfamily is involved in the metabolic activation of CP and AFB1. It has also been described that BIO is a potent inhibitor of CYP2B1/2 and a weaker inhibitor of CYP1A1. Therefore, antagonism of CP-and AFB1-induced mutagenic effects by BIO could have been mediated-at least in part-by the inhibition of CYP2B enzymes.     

Retinol (vitamin A) as a modifier of 2-aminofluorene and 2-acetyl-aminofluorene mutagenesis in the Salmonella/microsome assay

Vitamin A (retinol) has been demonstrated to modify the mutagenic activity of the aromatic amines, 2-aminofluorene (2AF) and 2-acetylaminofluorene (2AAF) when added to the Ames Salmonella/mammalian microsome assay. Low amounts of retinol (2–20 g/plate) increased the mutagenicity of both 2AF and 2AAF. At higher doses (50–150 g/plates) the mutagenicity of 2AAF remained unchanged while the mutagenicity of 2AF gradually decreased.The present data do not support the hypothesis that retinol generally acts as an inhibitor of in vitro metabolic activation of procarcinogens.Abbreviations 2AF 2-aminofluorene - 2AAF 2-acetylaminofluorene     

Potential pitfalls associated with testing of enzyme preparations in the Salmonella/microsome assay

The effect of a sample of food enzyme preparations on S9 activity was evaluated in bacterial mutation assays with the Salmonella typhimurium strains TA98 and TA100 using benzo(a)pyrene, 2-aminoanthracene and 2-aminofluorene as model compounds. Under the experimental conditions applied, Aspergillus oryzae protease and porcine pancreas trypsin, applied at low non-toxic doses, proved to effectively inhibit the metabolic activation of benzo(a)pyrene by Aroclor induced rat liver 9, while the activation of 2-aminoanthracene and 2-aminofluorene was only marginally affected. The tolerance of metabolic activation of 2-aminoanthracene to the presence of proteolytic enzymes, compared to the strong inhibition elicited on the metabolic activation of benzo(a)pyrene, points to the involvement of different components of liver S9 in their biotransformation. Overall, data indicate that the use of 2-aminoanthracene as positive control in the Ames test can give a misleading indication of S9 proficiency, and thus it should be used with caution or in conjunction with other chemicals, especially in the testing of crude enzyme preparations in which proteases may be present as minor components.     

Detection of genotoxicity in fried bacon by the Salmonella/mammalian microsome mutagenicity assay

The potential for mutagen formation in fried bacon and the possible reduction or elimination of this hazard was examined in the Salmonella/mammalian microsome mutagenicity assay using Salmonella typhimurium strain TA98. Alkaline dichloromethane extracts were prepared from green pork bellies, commercial bacon (nitrite-treated and nitrite-free), and pilot-plant bacon (nitrite-free). When fried, all forms of bacon and the green belly samples gave positive mutagenic responses with the plate-incorporation technique. Unfried samples were not mutagenic. Aroclor-activated rat-liver S-9 fractions plus NADPH were essential to demonstrate a mutagenic response. When the frying temperature was held constant (171°C) maximum mutagen formation was observed in samples fried for 6 min; when samples were fried for 6 min a mutagenic response which increased with temperature, in a linear manner, was observed at temperatures above 125°C. Volatile nitrosamines were not detected in the bacon samples. The data indicate the generation of one or more mutagens in fried bacon and green pork belly, the levels of which can be reduced by decreasing heating temperature and/or time.     

In vitro mutagenicity assessment of aluminium oxide nanomaterials using the Salmonella/microsome assay

The aim of the current study was to evaluate the potential mutagenicity of aluminium oxide nanomaterials (NMs) (Al₂O₃-30 nm and Al₂O₃-40 nm). Characterization of the NMs was done before the initiation of the study. The mutagenicity of the NMs was studied by the Ames test with Salmonella typhimurium TA100, TA1535, TA98, TA97a and TA102 strains, in the presence and absence of the S9 mixture. Based on a preliminary cytotoxicity study conducted on the strains, different concentrations of Al₂O₃-30 nm, Al₂O₃-40 nm and Al₂O₃-bulk were selected. At all the concentrations tested, Al₂O₃-30 nm and Al₂O₃-40 nm did not significantly increase the number of revertant colonies compared to the Al₂O₃-bulk and control with or without S9 mixture. Our findings suggest that Al₂O₃ NMs were devoid of any size and concentration dependent mutagenicity compared to the Al₂O₃-bulk and control.     

Comparative mutagenicity tests in the Salmonella/microsome assay with rat and woodchuck S9 preparations

The Salmonella mutagenicity assay was utilized to compare the hepatic S9 fractions from untreated and 3-methylcholanthrene (MC) induced woodchucks with Aroclor 1254 induced rats. Three known promutagens, benzo[a]pyrene (BP), 7,12-dimethylbenz[a]anthracene (DMBA), and 2-aminofluorene (AF) were tested at 5 concentrations with the strain TA100 against 3 levels of S9 fraction. Both woodchuck S9 fractions were as effective as the rat S9 in activating BP and both were more effective than the rat S9 in activating DMBA. Untreated woodchuck S9 was also as effective as rat S9 in activating AF. The protein content of the S9 fraction did not differ significantly between rats and woodchucks, but the P-450 content of the rat S9 was approximately 3.5 times that of woodchuck.     

Mutagenicity evaluation of riot control agent o-chlorobenzylidene malononitrile (CS) in the Ames Salmonella/microsome test.

o-Chlorobenzylidene malononitrile (CS), a riot control agent, was evaluated for its possible mutagenic activity in the Ames Salmonella/mammalian microsome mutagenicity test. Five histidine-deficient (His-) mutant tester strains of Salmonella typhimurium--TA97a, TA98, TA100, TA102 and TA104--were used. The liquid preincubation procedure was used with metabolic activation (presence of S9 mixture) and without metabolic activation (absence of S9 mixture). For the experiments with metabolic activation, three different concentrations of S9 fraction (supernatant of Aroclor 1254-induced rat liver homogenate at 9000 g)--5%, 15% and 30% in S9 mixture--were used. Along with mutagenic activity, CS was also evaluated for cytotoxic activity in all the five tester strains of Salmonella typhimurium, both in the presence and absence of S9 mixture. The mutagenic and cytotoxic activities of CS were assessed by counting the His+ revertant colonies and by counting the microcolonies (His-, auxotrophs in the background lawn), respectively, and the respective mean values were compared with the relative negative (solvent) control. A dose range of 12.5-800 micrograms plate-1 for CS did not induce a mutagenic response either in the presence or absence of S9 mix. No change in the negative mutagenic response of CS has been observed even in the presence of an elevated level of S9 fraction in the S9 mix. A dose of 200 micrograms plate-1 for CS was found to be cytotoxic by decreasing the surviving cells as well as His+ revertant colonies; however, the effect was reduced in the presence of an elevated level of S9 fraction in the S9 mix.     

Terpenoid biotransformation in mammals. IV Biotransformation of (+)-longifolene, (-)-caryophyllene, (-)-caryophyllene oxide, (-)-cyclocolorenone, (+)-nootkatone, (-)-elemol, (-)-abietic acid and (+)-dehydroabietic acid in rabbits

The metabolism of (+)-longifolene, (-)-caryophyllene, (-)-caryophyllene oxide, (-)-cyclocolorenone, (+)-nootkatone, (-)-elemol, (-)-abietic acid and (+)-dehydroabietic acid was studied in rabbits. Each of these sesquiterpenoids was converted to primary, secondary or tertiary alcohols, among which the primary alcohol was predominant. A vinylic methyl group and an exomethylene group were easily hydroxylated and converted to a glycol via an epoxide in many cases. Eight new metabolites were determined by chemical and spectroscopic methods.     

Mutagenicity evaluation of azaperone in the Salmonella/microsome test

Azaperone was evaluated for its mutagenic potential by the Salmonella/microsome test. No mutagenic activity towards six S. typhimurium strains could be evidenced with azaperone at doses up to 2,000 micrograms/plate, either without or with metabolic activation at usual test conditions. Higher concentrations of liver post-mitochondrial fraction from Aroclor 1254 (ARO)-pretreated rats did not reveal any increase in the number of revertants towards S. typhimurium strains TA1537, TA1538 and TA98. Moreover, a plate-incorporation test with liver post-mitochondrial fractions from mice pretreated with phenobarbital (PB) and a liquid preincubation test with liver post-mitochondrial fractions from rats pretreated with ARO also failed to reveal any mutagenic action of azaperone towards S. typhimurium strain TA98. Thus, none of the tests used provided any indication of azaperone having a mutagenic action.     

Terpenoid biotransformation in mammals IV Biotransformation of (+)-longifolene, (-)-caryophyllene, (-)-caryophyllene oxide, (-)-cyclocolorenone, (+)-nootkatone, (-)-elemol, (-)-abietic acid and (+)-dehydroabietic acid in rabbits

1. The metabolism of (+)-longifolene, (-)-caryophyllene, (-)-caryophyllene oxide, (-)-cyclocolorenone, (+)-nootkatone, (-)-elemol, (-)-abietic acid and (+)-dehydro-abietic acid was studied in rabbits.

2. Each of these sesquiterpenoids was converted to primary, secondary or tertiary alcohols, among which the primary alcohol was predominant.

3. A vinylic methyl group and an exomethylene group were easily hydroxylated and converted to a glycol via an epoxide in many cases.

4. Eight new metabolites were determined by chemical and spectroscopic methods.     

Formulation,characterization, cytotoxicity and Salmonella/microsome mutagenicity (Ames) studies of a novel 5-fluorouracil derivative

5-Fluorouracil is one of the first line drugs for the systemic therapy of solid tumors like breast, colorectal, oesophageal, stomach, pancreatic, head and neck.It could be shown that sugars can improve the absorption across cell membranes and can help to bypass some pharmacokinetic problems. Carbohydrates as most common organic molecules are an important issue of plant and animal metabolisms. They are non toxic and have important duties in the body like participating in DNA and RNA synthesis and being responsible for energy production. In addition, they have many hydroxyl, aldehyde and ketone groups that attract attention for synthesis as a potential drug derivative. 1,2,3,-Triazole compounds have also important role in heterocyclic chemistry because of their pharmaceutical properties and their high reactivity, which could be used as a building block for complex chemical compounds. In this study, following the “Click Reaction” of 5-FU and tetra-O-acetylglycose the 5-fluorouracil derivative 1-[{1′-(2″,3″,4″,6″-tetra-O-acetyl-β-d-glycopyronosyl)-1′H-1′,2′,3′-triazole-4′-yl} methyl]5-fluorouracil was synthesized.Following, a micellar formulation of 5-Fluorouracil derivative was prepared and characterized in terms of particle size, polydispersity index, zeta potential, refractive index and pH. Furthermore, the cytotoxicity and mutagenicity of the 5-fluorouracil derivative was investigated using an in vitro cell culture model and the AMES test. According to the results of this study, the novel 5-fluorouracil derivative could be a drug candidate for the therapy of cancer and needs further in vivo investigations.     

Studies on the genotoxic properties of essential oils with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay

Genotoxic properties of essential oils from Anthemis nobilis L., Artemisia dracunculus L., Salvia officinalis L., Salvia sclarea L., Satureja hortensis L., Satureja montana L., Thymus capitatus L., Thymus citriodorus Schreb., Thymus vulgaris L., Citrus bergamia Risso, were studied with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay. The essential oil of Artemisia dracunculus L. "Piemontese" turned out to be active in the rec-assay but not in the Salmonella test. DNA-damaging activity was demonstrated to be due to the estragol component of the oil. Advantages of the combined use of these two short-term microbial assays in genotoxic studies are discussed.     

Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): IX. Evaluation of the mutagenic potential of synthesized L-valyl-L-prolyl-L-proline in the Salmonella-Escherichia coli/microsome, incorporation assay

The objective of this study was to assess the mutagenic potential of a synthesized tripeptide, L-valyl-L-prolyl-L-proline (VPP), to induce mutational changes in Salmonella typhimurium LT2 strains TA1535, TA1537, TA98, and TA100, and Escherichia coli strain WP2uvrA in the classical Ames test protocol. Bacteria were exposed to plate concentrations of VPP of 0, 156.2, 312.5, 625, 1250, 2500, and 5,000 microg/plate in distilled water, in the presence and absence of Aroclor 1254-induced rat liver homogenate preparation (S9). Positive-control agents included sodium azide (TA100 and TA1535); 2-aminoanthracene (TA98, TA100, TA1535, TA1537, and WP2uvrA); 9-aminoacridine (TA1537); 2-nitrofluorene (TA98); and N-ethyl-N'-nitro-N-nitrosoguanidine (WP2uvrA) in DMSO. Incubations were conducted at 37 degrees C for about 48 h then revertant colonies were counted. All positive-control agents were consistently and unequivocally positive, but there was no evidence that VPP induced increases in the incidences of revertant colonies in any bacterial strain with and without metabolic activation. These findings were replicated in a second, confirmatory test performed with and without S9. The results of the experiments revealed no treatment-associated changes in the incidence of revertant colonies in any bacterial strain tested. These results support a conclusion that, under the experimental conditions described, there is no evidence that VPP possesses mutagenic potential.     

Upper airway and pulmonary effects of oxidation products of (+)-alpha-pinene,d-limonene,and isoprene in BALB/c mice

The oxidation products (OPs) of ozone and the unsaturated hydrocarbons d-limonene, (+)-alpha-pinene, and isoprene have previously been shown to cause upper airway irritation in mice during 30-min acute exposures. This study evaluated the effects of OPs and the hydrocarbons themselves on the upper airways, the conducting airways, and the lungs over a longer exposure period. The time course of development of effects and the reversibility of effects were investigated; in addition, we assessed possible exacerbation of sensory responses of the airways to the unreacted hydrocarbons. Respiratory parameters in male BALB/c mice were monitored via head-out plethysmography. Exposures to OPs or hydrocarbons were for 60 min, followed by a 30-min challenge period with air or hydrocarbon, and a 15-min recovery period with air only. Experiments were also performed where limonene/ozone exposures were separated 6 h from the challenge period. Ozone concentration in the reaction mixture was 3.4 ppm, and concentrations of hydrocarbons were 47 ppm (alpha-pinene), 51 ppm (d-limonene), and 465 ppm (isoprene). Due to reaction, the ozone concentration at the point of exposure was less than 0.35 ppm; exposure to 0.30 ppm ozone for 60 min did not produce effects different from air-exposed control animals. As previously established, upper airway irritation was a prominent effect of OP exposure. In addition, over the longer exposure period we observed the development of airflow limitation that persisted for at least 45 min postexposure. All effects from limonene/ozone exposures were reversible within 6 h. Exposures to OPs did not cause enhanced upper airway irritation during challenge with the hydrocarbons, indicating that a 1-h exposure to OPs did not increase the sensitivity of the upper respiratory system. However, airflow limitation was exacerbated in animals exposed to d-limonene alone immediately following exposure to limonene OPs. These findings suggest that terpene/ozone reaction products may have moderate-lasting adverse effects on both the upper airways and pulmonary regions. This may be important in the context of the etiology or exacerbation of lower airway symptoms in office workers, or of occupational asthma in workers involved in industrial cleaning operations.     

Effects of (+/-)- (+)- and (-)-metoprolol, (+/-)- (+)- and (-)-pindolol, (+/-)-mepindolol and (+/-)-bopindolol on the rat left atria and portal vein.

1. The effects of (+/-)- (+)- and (-)-metoprolol, (+/-)- (+)- and (-)-pindolol, (+/-)-mepindolol and (+/-)-bopindolol on the beta 1-adrenoceptor mediated responses of the rat left atria and the beta 2-adrenoceptor mediated responses of the rat portal vein to isoprenaline have been determined. 2. Racemic and (-)-metoprolol were selective beta 1-adrenoceptor antagonists. (+)-Metoprolol was devoid of beta-adrenoceptor antagonistic activity. 3. Racemic and (-)-pindolol were potent and (+)-pindolol was a modest beta-adrenoceptor antagonist. 4. (+/-)-Mepindolol and (+/-)-bopindolol were apparently competitive antagonists of the isoprenaline beta 1-adrenoceptor mediated responses of the rat left atria but non-competitive antagonists of the isoprenaline beta 2-adrenoceptor mediated responses of the rat portal vein. 5. It is suggested that (+/-)-mepindolol and (+/-)-bopindolol are slowly dissociating beta-adrenoceptor antagonists and the non-competitive antagonism can only be detected on tissues with modest receptor reserves for maximum responses to isoprenaline.     

Conformational analysis and structural comparisons of (1R,3S)-(+)- and (1S,3R)-(-)-tefludazine, (S)-(+)- and (R)-(-)-octoclothepin, and (+)-dexclamol in relation to dopamine receptor antagonism and amine-uptake inhibition

Conformational analysis with molecular mechanics (MM2(85] and molecular superimposition studies of (1R,3S)-(+)- and (1S,3R)-(-)-4-[3-(4-fluorophenyl)-6-(trifluoromethyl)indan-1-yl]-1- piperazineethanol (tefludazine) and (S)-(+)- and (R)-(-)-octoclothepin have been employed to identify biologically active conformations of these compounds with respect to dopamine receptor antagonism and amine-uptake inhibition. In contrast to what is commonly assumed, these studies indicate that the conformation of (S)-(+)-octoclothepin responsible for the dopamine receptor antagonism is different from the one observed in the crystal. From least-squares molecular superimpositions with the potent and stereoselective dopamine receptor antagonist (1R,3S)-tefludazine, biologically active conformations for the two compounds on the dopamine receptor have been deduced. This analysis also rationalizes the enantioselectivity of octoclothepin on the dopamine receptor. The X-ray structure of (S)-(+)-octoclothepin is shown to correspond structurally to the 1S,3R enantiomer of tefludazine, which is an amine-uptake inhibitor. This correspondence provides a structural basis for the norepinephrine (NE) uptake blocking properties of octoclothepin. It is predicted that the enantioselectivity of the NE-uptake inhibition of octoclothepin should be low with the S-(+) enantiomer as the more active optical isomer. A comparison of the deduced biologically active conformation of (S)-(+)-octoclothepin with (+)-dexclamol is also discussed on the basis of earlier derived superimposition studies with (+)-dexclamol.     

Genotoxicity assessment of NIM-76 and its formulation (pessary) in an in vitro Ames Salmonella/microsome assay and in vivo mouse bone marrow micronucleus test

Abstract

The possible genotoxic potential of NIM-76, a volatile fraction obtained from neem oil, having promising contraceptive activity, as well as its formulation product, called pessary (7.5% NIM-76 in polyethylene glycol), were evaluated in the Ames assay and mouse bone marrow micronucleus (MN) assay. Genotoxicity of NIM-76 (0.1–1000?µg/plate) and pessary (0.1–10?000?µg/plate) were studied using the liquid preincubation protocol of the Ames assay both in the presence and absence of S9. Likewise, the ability of NIM-76 [1–1000?mg/kg body weight (b.w.)] and its formulation product (18.75–300?mg/kg b.w.) to induce clastogenic effects were studied in the female mouse bone marrow MN test by using a two-dose intraperitoneal treatment protocol. There was no increase in the number of revertant colonies resulting from NIM-76 or pessary at any of their doses over the respective negative control plates, either in the presence or absence of S9. Similarly, in the MN assay, neither of them showed any clastogenic activity because there was no significant increase in the frequency of micronucleated polychromatic erythrocytes, over the negative control group of animals. The use of this compound in humans is therefore not likely to have mutagenic effects and may be considered as safe with regard to genotoxic potential.