Repellency to ticks (Acari: Ixodidae) of extracts of Nigella sativa (Ranunculaceae) and the anti-inflammatory DogsBestFriend™.

Motivated by observations that the canine anti-inflammatory cream DogsBestFriend™ (DBF) appeared to deter flies, mosquitoes, and ticks from treated animals, repellent efficacy bioassays using four species of ticks were conducted with three extracts of Nigella sativa L. (Ranunculaceae), a constituent of DBF. The DBF cream was tested against nymphs of lone star tick, Amblyomma americanum (L.). In vertical filter paper assays, the three extracts applied at 0.413 mg extract/cm(2) filter paper repelled 96.7-100 % of brown dog tick, Rhipicephalus sanguineus (Latreille) nymphs, whereas, at the same rate, only one extract repelled >90 % A. americanum nymphs. Adult (mixed sexes) American dog ticks, Dermacentor variabilis (Say), required a higher concentration to be repelled effectively; two extracts, applied at 0.827 mg extract/cm(2) filter paper, repelled ≥90 % of the D. variabilis. In contrast, all extracts applied at much lower concentration (0.206 mg extract/cm(2) filter paper) repelled 100 % adult blacklegged ticks, Ixodes scapularis Say (only females tested). Of the two more repellent extracts, one lost most of its activity against A. americanum nymphs in <4 h when applied at 0.827 mg extract/cm(2) filter paper, whereas the other repelled 66.7 % of the nymphs at 192 h after application. At 0.206 mg extract/cm(2) filter paper, one extract was as repellent as deet against A. americanum nymphs. In a vertical bioassay in which nylon organdy was substituted for filter paper, DBF, at the rates of 1.67 and 0.835 mg cream/cm(2), repelled 76.7 and 30.0 % A. americanum nymphs, respectively. These findings indicate that when applied appropriately DBF should afford some protection to canines against tick bites.

Gastric mucosal cell model for estimating relative gastrointestinal toxicity of non-steroidal anti-inflammatory drugs.

The study objective was to characterize the AGS human gastric mucosal cell line as a model for estimating gastrointestinal toxicity of COX-inhibiting compounds. Rofecoxib, celecoxib, nimesulide, ibuprofen, indomethacin, aspirin, salicylic acid, naproxen and acetaminophen were tested for inhibition of COX-2-mediated prostaglandin E2 synthesis in A549 and AGS cells. The IC50 ratio AGS/A549 was calculated as an estimate of the therapeutic index (TI) for gastrointestinal toxicity. Calculated IC50 values of non-steroidal anti-inflammatory drugs (NSAIDs) in A549 cells were in excellent agreement with published values (r = 0.996; P < 0.005). Calcium ionophore induction of arachidonic acid release in AGS cells provided TI similar to those using platelets and A549 cells (r = 0.918; P < 0.01). The AGS/A549 model exhibited lower TI than the platelet/A549 model. Spearman ranking correlated clinical NSAID gastropathy with lower AGS TI values. The AGS cell line has excellent potential to serve as a model for assessing the gastrointestinal effects of COX-inhibiting compounds.

Discordant expression of the cyclin-dependent kinases and cyclins in rat liver following acute administration of the hepatocarcinogen [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (WY14,643).

Cellular proliferation is an essential aspect of chemical carcinogenesis. At the core of cell cycle regulation is a family of serine/threonine protein kinases termed cyclin-dependent kinases (cdk). Cdk activity, which directs progression through the cell cycle, is dependent upon cdk binding to the appropriate, phase-specific cyclin proteins. Alterations in hepatic cdk1, cdk2, cdk4, cdk5, and cyclin protein expression were determined in response to acute dosing of the prototypic peroxisome proliferator and hepatocarcinogen [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (WY14,643). Intraperitoneal dosing of 45 mg WY14,643/kg daily for 4 days to young, male rats produced dramatic increases in hepatic protein expression of all cdk analyzed as well as cyclins B, D2, D3, and proliferating cell nuclear antigen (PCNA). The largest relative increases, 6.1-, 2.8-, 11-, 83-, and 7.9-fold, were seen with cdk1, cdk4, cyclin B, cyclin D3, and PCNA, respectively. Increases of only 1.8-, 2-, 1.6-, and 1.4-fold were noted, respectively, for cdk2, cdk5, cyclin D2, and cyclin E. Analysis of gel filtration fractions indicated that PCNA co-eluted with cdk1 from the WY14,643-treated rats as a 70-80 kDa molecular complex. In contrast, cdk4, cdk5 and D cyclins migrated as much larger complexes with an estimated MW of approximately 180-190 kDa.

Effect of diet on the hepatotoxicity of polybrominated biphenyls (FireMaster PB-6).

The consumption of diets formulated with Cruciferae vegetables, e.g., cauliflower, cabbage, and Brussels sprouts, has been shown to result in a stimulation of the intestinal and hepatic microsomal enzyme systems in rats. This study was designed to determine if this increase in intestinal and hepatic microsomal enzyme activity affected the hepatic response to polybrominated biphenyls (PBB). After three weeks of consuming either a semipurified or 25% cauliflower leaf-supplemented diet (CLD), male Sprague-Dawley rats were maintained for an additional 20 days on their respective diets containing either 0, 1, or 50 ppm PBB. A significant decrease in body weights, but not feed efficiency, was observed over all levels of PBB in animals consuming CLD compared to semipurified diets; consumption of up to 50 ppm of PBB had no effect on body weights with either diet. Relative liver weights (RLW), hepatic aryl hydrocarbon hydroxylase (AHH), N- and O-demethylase, as well as intestinal AHH were all increased in CLD-consuming animals before the addition of PBB. While PBB supplementation alone resulted in increased RLW, hepatic AHH, N- and O-demethylase, microsomal protein, and cytochrome P-450, rats consuming cauliflower diets + PBB had even higher RLW and N- and O-demethylase activity and microsomal protein concentrations. Hepatic PBB residue and total hepatic lipids were significantly reduced in CLD groups receiving 50 ppm PBB. These results suggest that the antitoxic effects of certain vegetables are related to more rapid metabolism and excretion of xenobiotic compounds.

Salmonella typhimurium (TA100) mutagenicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and its open- and closed-ring analogs.

The mutagenicities of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX, compound 1), 3-chloro-4-(dichloromethyl)-2(5H)-furanone (RMX, compound 6), and 2-(dichloromethyl)-3,3-dichloropropenal (TCB, compound 7) were determined in the same assay and in repetitive determinations using Salmonella typhimurium (TA 100) without microsomal fraction activation. In addition, the mutagenicity of 2-methyl-3,3-dichloropropenal (compound 8) was assayed in the same manner although not simultaneously with MX, RMX, and TCB. This study was undertaken to ascertain the role of open- and closed-ring forms of MX in the mutagenicity of MX. MX proved to be roughly 100 times more mutagenic than the open-ring analogue TCB and 10 times more mutagenic than the closed-ring analogue RMX. Compound 8 was inactive. Assay stability of the three active compounds in Vogel-Bonner medium at 38 degrees C was estimated as the chemical half-life values by following the change in UV absorbance at selected wave lengths. Half-life values were 10.7, 2.6, and 2.8 hr, respectively, for MX, RMX, and TCB. The enhanced mutagenicity of MX relative to RMX and TCB is attributed to the intrinsic mutagenicity of MX and its greater stability is judged to play only a minor role. Moreover, the greater mutagenicity of the closed-ring analogue RMX relative to the open-ring analogue TCB points to the ring form of MX as the active species even though the open form of MX is predominant under assay conditions.

In vitro activation of the promutagens 2-acetamidofluorene, cyclophosphamide and 7,12-dimethylbenzanthracene by constitutive ferret and rat hepatic S-9 fractions.

The ability of the ferret to metabolically activate promutagenic compounds was compared with that of the rat, using the Salmonella/microsome assay. Three compounds which require biotransformation to mutagenic metabolites, 2-acetamidofluorene (2-AAF), cyclophosphamide (CPA), and 7,12-dimethylbenzanthracene (DMBA), were studied. Metabolic activation was provided by ferret or rat hepatic S-9 fractions at 5 levels for each chemical, and optimal S-9 levels as well as dose-response curves were obtained. Interspecies mutagenic activity was quantitated on the basis of mg liver, mg S-9 protein, and nmoles P-450. The slopes of the dose-response curves and the lowest chemical dose required for a significant response were also compared. Although constitutive levels of rat hepatic cytochrome P-450 were shown to be higher than those of the ferret, in vitro mutagenic activation by ferret S-9, at S-9 levels which caused activation in both species, was greater than or equivalent to activation by rat S-9 for these chemicals, based on all parameters studied. The results showed that the equilibrium between activation and detoxification reactions is dependent upon the chemical dose and S-9 level present.

In vitro activation assays with hepatic S9 preparation from wild and laboratory reared woodchucks in the Salmonella mutagenicity test.

The Salmonella mutagenicity assay was utilized to compare hepatic S9 fractions derived from wild and laboratory reared woodchucks (Marmota monax). Two promutagens, 7,12-dimethylbenz[a]anthracence (DMBA) and 2-amino-fluorene (AF) were tested at 5 concentrations with the tester strains TA98 and TA100, against 2 levels of S9 fraction. AF produced similar number of revertants with the S9 fraction from wild and laboratory-reared animals. DMBA produced 2-4 times more revertant colonies at 50 microliter S9/plate with wild woodchuck S9 than with S9 from the laboratory-reared animals with both tester strains. It was concluded that natural inducers in the wild woodchuck diet may have contributed to the increased reversion frequency over laboratory reared woodchucks. Dose-response parameters for the activation of DMBA by S9 fraction from woodchucks and rats were compared with TA100. Woodchuck S9 had 3-40 more revertants/nmol and a 100-fold lower threshold of response than S9 from Aroclor 1254-induced rats.

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.

Benzo[e]pyrene elicits changes in the biochemical activities and chromatographic behavior of murine hepatic cytochromes P-450 that are distinct from those induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The objective of this study was to examine the potential for a specific ligand of carcinogen binding protein (CBP) to induce changes in the overall character of hepatic microsomal cytochromes P-450 (P450) and to compare potential changes with those induced by an Ah receptor ligand. Benzo[e]pyrene (BeP) was previously shown to bind CBP with high affinity and Ah receptor with low affinity. In contrast, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binds Ah receptor avidly and CBP weakly. Hepatic microsomes were prepared from C57BL/6J (B6) and DBA/2J (D2) mice treated with corn oil, BeP or TCDD. Relative to corn oil controls, pretreatment of B6 mice with BeP or TCDD increased the nmol P450/mg microsomal protein content 26 and 28%, respectively. In D2 mice, nmol P450/mg microsomal protein was increased 23% in the BeP pretreatment, while TCDD pretreatment had no effect relative to the corn oil controls. For the O-alkyl ethers of resorufin, rates of metabolism (per nmol P450) were affected differently in B6 and D2 by BeP pretreatment. Pentoxyresorufin O-dealkylase activity was reduced to 44% of control activity in B6 mice and increased 39% relative to controls in D2 mice. BeP pretreatment had no effect on ethoxyresorufin O-dealkylase activity in B6 mice, while this activity was decreased to 58% of controls in D2 mice. Additionally, benzyloxyresorufin O-dealkylase activity was reduced to 65% of control levels in B6 mice and not affected in D2 mice. Methoxyresorufin O-dealkylase activity was reduced in both strains to an average of 55% of control values. As expected, TCDD pretreatment resulted in increases of all O-dealkylations measured in both strains of mouse. For both inbred strains of mouse, anion exchange chromatography revealed a P450 peak associated with BeP pretreatment that was not present in chromatograms generated with corn oil or TCDD pretreatments. Results of enzyme linked immunosorbant assays also indicated that the pattern of P450 isoenzyme expression associated with BeP pretreatment was distinct from that associated with TCDD pretreatment. Overall, these data show that treatment with a specific ligand of CBP induces changes the biochemical activities and chromatographic behavior of P450 isozymes in murine hepatic microsomes. Moreover, they indicate that changes in P450 occurring after treatment with a CBP ligand are distinct from those changes that are associated with treatment with an Ah receptor ligand (TCDD). Differences between B6 and D2 strains suggest that the hepatic P450 changes occurring in response to pretreatment with a CBP ligand may be influenced by the presence of Ah receptor.

Effects of ciprofloxacin and enrofloxacin on zoxazolamine kinetics, plasma concentration and sleeping times in mice.

The treatment of CD1 male mice with either ciprofloxacin (CP) or enrofloxacin (EF) prior to zoxazolamine (ZX) administration increased the mean ZX sleeping times to, respectively, 162 and 156% of the control (ZX alone). At the end of the sleeping time, the mean ZX plasma concentration in controls was 27.2 micrograms/ml and was not different in EF- or CP-treated groups (87% and 95% of controls, respectively). The animals coadministered with CP or EF and ZX eliminated the latter more slowly than the controls. The estimated zero-time drug concentration of the disposition curves of both the CP- and EF-treated groups as well as the apparent half-life of elimination and apparent overall rate of elimination of the CP-treated group were different from the control values.

The interaction of L-triiodothyronine and 2,3,7,8-tetrachlorodibenzo-p-dioxin on Ah-receptor-mediated hepatic Phase I and Phase II enzymes and iodothyronine 5'-deiodinase in thyroidectomized rats.

Across all levels of L-triiodothyronine (L-T3) treatment, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in increased hepatic cytochrome P-450-associated activities of 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-dealkylase (EROD) and aryl hydrocarbon hydroxylase (AHH). The treatment of thyroidectomized rats with L-T3 at physiologic replacement levels in concert with TCDD produced an increase in ECOD, EROD and AHH activity above that seen with only TCDD. TCDD as well as L-T3 enhanced the activity of hepatic 1-naphthol glucuronyl transferase (NGT). In addition, the combined effect of L-T3 and TCDD resulted in similar levels of induction of NGT at both physiologic and supraphysiologic doses of L-T3. TCDD treatment resulted in elevated serum T3 levels at both physiologic and supraphysiologic levels of L-T3. One TCDD dose inhibited hepatic microsomal 3,3',5'-triiodothyronine (reverse T3) 5'-deiodinase activity by 61% in thyroidectomized, T3-untreated rats. The inhibition of 5'-deiodinase activity was partially overcome by increasing the T3 dose.

Benzo[e]pyrene pretreatment of immature, female C57BL/6J mice results in increased bioactivation of aflatoxin B1 in vitro.

Hepatic microsomes were prepared from immature C57BL/6J mice 24 h after receiving intraperitoneal injections of either corn oil, benzo[e]pyrene (BeP, 50 mg/kg) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 4 x 10(-3) mg/kg). The capacity of these hepatic microsomes to bioactivate aflatoxin B1 (AFB1), 2-aminoanthracene (AA), benzo[a]pyrene (BaP), 3-methylcholanthrene (MC), 7,12-dimethylbenzanthracene (DMBA), BeP and pyrene (PY) was measured using strain TA100 in the Salmonella typhimurium/microsome reversion assay. BeP pretreatment of mice resulted in a 33% increase in mutagenic potency (MP) of AFB1 over the corn oil controls and a 70% increase in MP relative to TCDD-pretreated microsomes. With AA, BaP and DMBA as promutagens, BeP pretreatment reduced MP an average of 24%, while TCDD pretreatment increased MP of these 3 promutagens 263% compared to controls. Since the general effects of BeP and TCDD on murine hepatic cytochrome P-450 (P450)-mediated activities in this study were discordant, it appears that changes in P450 activity by BeP pretreatment are not mediated through the Ah receptor.

Testing of 2,4,5-T-amino acid conjugates for mutagenic activity in Salmonella typhimurium strains.

Since amino acid conjugates are plant metabolites of the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 5 amino acid conjugates (aspartic acid, glutamic acid, leucine, methionine and tryptophan) of 2,4,5-T were tested for possible mutagenic activity utilizing 5 strains of Salmonella typhimurium (TA97, TA98, TA100, TA1535 and TA1538) with and without rat-liver microsomal and cytosolic enzymes. These compounds did not cause any significant increase in reversions when compared with controls in the presence or absence of the activating system. Further, linear regression analysis showed no significant (p less than 0.05) dose-response relationships. Thus, it was concluded that the tested amino acid conjugates of 2,4,5-T are not mutagens or promutagens in these assays.

A cell culture analogue of rodent physiology: Application to naphthalene toxicology.

The difficulties of large-scale animal testing of compounds has spurred development of in vitro testing methods and physiologically based pharmacokinetic models (PBPK). In existing in vitro methods, tissue interactions occurring in vivo are not reproduced accurately and in PBPKs the a priori prediction of metabolism is difficult. Through development of a multicompartmental, multiple cell type bioreactor system these limitations can be circumvented. A cell culture analogue (CCA) of a PBPK was developed. The CCA contains multiple chambers, each of which represents a tissue or group of similar tissues as specified in the PBPK. Proof-of-concept experiments were done using naphthalene as a model. Naphthalene is converted into naphthalene oxide and the circulation of this reactive metabolite from the liver to lung is a possible mechanism for lung injury. A CCA with liver, lung and other tissue compartments was constructed. This system was used in conjunction with cultured H4IIE rat hepatoma cells and L2 rat lung cells to study the importance of circulated naphthalene metabolites (presumably naphthalene oxides) on lung cell toxicity in rodents. By increasing the number of cells and/or inducing cytochrome P-450 activity in the liver compartment, lung cell mortality was increased. Glutathione depletion in the lung and liver cells was also observed. These results indicate that the CCA is a potentially useful concept for studying the action of compounds with reactive metabolites.

Modification of beet and cabbage diets of aflatoxin B1-induced rat plasma alpha-foetoprotein elevation, hepatic tumorigenesis, and mutagenicity of urine.

Weanling male Fischer rats were fed a purified diet or diets containing 25% (w/w) freeze-dried ground beets or cabbage with or without 1 ppm aflatoxin B1 (AFB1) for 26 wk. In 3-7 wk the cabbage diet diminished, while the beet diet enhanced AFB1-induced plasma alpha-foetoprotein (AFP) elevation. When the experiment was extended to 42 wk by maintaining the animals on the purified (basal) diet for a further 16 wk the rats that had consumed AFB1 in the beet diet had 72 +/- 14 tumours/liver (mean surface diameter of tumours, 6.13 +/0 4.69 mm); animals that had been given AFB1 in the control diet had 30 +/- 16 tumours/liver (mean surface diameter, 4.36 +/- 3.16 mm); rats that had been given AFB1 in the cabbage diet had 13 +/- 5 tumours/liver (mean surface diameter, 4.28 +/- 2.89 mm). In the Salmonella/mammalian microsomal test, urine from rats fed AFB1 with beets caused significantly (P less than 0.05) more revertants in Salmonella typhimurium strain TA98 than did urine from rats fed AFB1 with purified or cabbage diets. The beet- and cabbage-containing diets had no effect on the plasma AFP concentration, hepatic tumorigenesis, or the mutagenicity of urine in rats receiving no AFB1. The evidence suggests that non-nutrient components of common vegetables may influence the response to chemical carcinogens, and that AFP determinations are useful in the rapid identification of dietary factors that modify carcinogenesis.

Subchronic feeding study of grape colour extract in beagle dogs.

The effect of feeding Welch's Special Grape Color Powder Type BW-AT at dose levels of 7.5 and 15% w/w in the diet for 90 days was studied in beagle dogs. Body-weight gain of male and female dogs at the high dose level was significantly decreased compared with control dogs. No other treatment-related effects were seen in food consumption, haematology, clinical chemistry, ophthalmology or gross and histopathological findings.

Reproduction study of grape colour extract in rats.

The effect of Welch's Special Grape Color Powder Type BW-AT on reproductive performance was studied through two generations of Sprague-Dawley rats and a subchronic study was carried out on the F1 animals. The grape colour powder at dietary levels of 7.5 and 15.0% (w/w) had no adverse effects on reproductive performance. Body weights for F0 and F1 generation pups at both dose levels were significantly lower (P less than 0.05) than those of control pups at 21 days after birth. During the 13-wk subchronic feeding study of F1 rats, the body-weight gain of female rats in the high-dose group was reduced compared with the controls (P less than 0.05). Food conversion data was comparable among groups, thus the decrease in body-weight gain during this phase was most likely the result of the lower calorific value (w/w) of the feed supplemented with the grape colour powder compared with the control feed. No toxic effects or pathological changes were noted in rats fed grape colour powder.

Reproduction and subchronic feeding study of carnauba wax in rats.

The reproductive performance of Wistar rats fed carnauba wax at levels of 0.1, 0.3 or 1% in the diet and the effects of subchronic administration of carnauba wax at these dose levels on the resultant progeny were studied. Reproductive indices, body-weight gain, food consumption, haematological and clinical chemical data, ophthalmic, gross and histopathological examinations were used to study the possible toxic or pathological effects. Serum free fatty acid levels were found to be decreased in male and female rats fed carnauba wax at dietary levels of 0.3 and 1.0%. No other effects of feeding carnauba wax at levels up to 1.0% of the diet were observed.

Subchronic feeding study of carnauba wax in beagle dogs.

Carnauba wax fed at levels of 0.1, 0.3 and 1% in the diet to beagle dogs for 28 wk did not produce evidence of toxicity or pathological effects. Body weight gain, food consumption, clinical chemical, haematological, and urine analysis data, and organ weights of animals fed carnauba wax were comparable with those of control animals. Ophthalmic, gross and histopathological examinations revealed no significant treatment-related findings.

Use of amprolium for the control of coccidiosis in pheasants.

Amprolium administered in feed during the first 4 weeks of life at a level of 0.0175% protected pheasants against three major pathogenic species of coccidia (Eimeria colchici, E. duodenalis, and E. phasiani) when they were exposed at 2 weeks of age. The difference was significant when mortalities were compared between medicated infected (3%) and unmedicated infected (35%) pheasants. The manufacturer's proposed level (0.0175%) and twice the proposed level (0.0350%) of amprolium had no significant effect on weight gains or mortality in the safety trial. Amprolium residues found in the muscles and livers of pheasants that received either level of amprolium did not exceed the tolerance levels for chickens and turkeys permitted by the U.S. Food and Drug Administration.