Search

The isolated Actinomycete, Streptomyces krainskii, SUK -5 was found to decolorize and degrade textile dye Reactive blue–59.This azo dye was decolorized and degraded completely by Streptomyces krainskii SUK–5 at 24 h in shaking condition in the nutrient medium at pH 8. Induction in the activity of Lignin Peroxidase,and NADH-DCIP Reductase and MR reductase represents their role in degradation .The biodegradation was monitored by TLC, UV vis spectroscopy, FTIR. and GCMS analysis. Microbial and phytotoxicity studies of the product were carried out.

The efficient cellulolytic microorganism was isolated from soil samples collected from Shivaji University campus. Biochemical test and phylogenetic analysis of isolated culture identified as Klebsiella sp. PRW-1. The isolated culture could utilize pure cellulosic substrates (carboxymethylcellulose (CMC) and avicel) and different agricultural wastes like sugarcane bagasse, sugarcane barbojo, sorghum husks, grass powder, corn straw and paddy straw by producing a large amount of endoglucanase, exoglucanase, β-glucosidase, filter paperase (FPU), xylanase and glucoamylase. The reducing sugar production was found higher in the presence of grass powder and sugarcane barbojo. Effects of different physic-chemical parameters to achieve maximum cellulolytic enzymes production were systematically investigated. The effects of supplementation of different metals additives as well as the stability in the presence of higher temperature, pH and commercial detergents, on cellulolytic enzymes were also studied. The foregoing result increases the applicability of the strain for the utilization and bioconversion of lignocellulosic biomass that could be used for bioenergy production. [ABSTRACT FROM AUTHOR]

Objective: To study the effect of sodium sulfadimethylpyrimidine (SDMP) on different forms of CYP 450 enzymes induced by phenobarbital (CYP 2B1, 2B2 and 3A), isoniazid (CYP 2E1), benzo(a)pyrene (CYP 1A1), clotrimazole (CYP 3A), and clofibrate (CYP 4A). Materials and Methods: Chickens (Hubbard, male) weighing 250-300 g were divided into 17 groups of six each. Five experimental sets were prepared containing three subgroups each to test five different inducers. Microsomes were isolated by calcium precipitation. The levels of electron transport components, CYP 450, cytochrome b5, and cytochrome creductase were determined using extinction coefficients. Activities of drug-metabolizing enzymes were assayed. Results: All inducers (phenobarbital, isoniazid, benzo(a)pyrene, clotrimazole, and clofibrate) showed significant induction of mixed function oxidase in chicken. The SDMP treatment of inducer-pretreated chicken caused a significant decrease in electron transport components and activities of drug-metabolizing enzymes when compared with treatment of inducer alone. Phenobarbital, isoniazid, and benzo(a)pyrene treatments of SDMP-pretreated chicken showed no significant change in induction pattern, however, significant alterations were observed in the induction pattern of clotrimazole and clofibrate. Conclusion: Our studies suggest that CYP 2B1, 2B2, 3A; CYP 2E1; CYP 1A1; CYP 3A and CYP 4A are susceptible species of CYP 450 to SDMP and its metabolites. The SDMP also affected in the induction pattern of some of the inducers with respect to CYP 450 isoforms. [ABSTRACT FROM AUTHOR]

Zinc oxide (ZnO) thin films have been successfully deposited onto fluorine doped tin oxide coated glass at substrate temperature of 400 °C and used as electrode in photoelectrocatalytic reactor. The untreated textile effluent was circulated through photoelectrocatalytic reactor under UVA illumination for the decolorization and degradation. Textile effluent was decolorized by 93% within 3h at room temperature with significant reduction in COD (69%). High performance liquid chromatography (HPLC) and Fourier transform infrared spectroscopy (FTIR) analysis of samples before and after decolorization confirmed the degradation of dyes molecules from textile effluent into simpler oxidizable products. Phytotoxicity study revealed reduction in toxic nature of textile effluent after treatment., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The nanocrystalline ZnO, TiO(2) and ZnO/TiO(2) films are deposited onto FTO-coated glass substrates by using spray pyrolysis technique. The structural, morphological, optical and photoelectrochemical properties of the ZnO, TiO(2) and ZnO/TiO(2) are investigated by X-ray diffraction, scanning electron microscopy, UV-Vis spectroscopy and photoelectrochemical techniques. XRD analysis shows that films are polycrystalline and having hexagonal and tetragonal crystal structure for pure ZnO and TiO(2). The photocatalytic degradation of methylene blue has been investigated with ZnO, TiO(2) and ZnO/TiO(2) photocatalysts. ZnO/TiO(2) thin films have proved quite effective mineralization of methylene blue, while pure ZnO and TiO(2) do not lead complete mineralization of methylene blue. The metabolites produced during degradation are analyzed by HPLC and Fourier transforms infrared spectroscopy. The by-products detected during degradation have been identified by GCMS technique., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Agrobacterium radiobacter MTCC 8161 completely decolorized the Crystal Violet with 8 hr (10 mg/L) at static anoxic conditions. The decreased decolorization capability by A. radiobacter was observed, when the Crystal Violet concentration was increased from 10 to 100 mg/L. Semi-synthetic medium containing 1% yeast extract and 0.1% NH4C1 has shown 100% decolorization of Crystal Violet within 5 hr. A complete degradation of Crystal Violet by A. radiobacter was observed up to 7 cycles of repeated addition (10 mg/L). When the effect of increasing inoculum concentration on decolorization of Crystal Violet (100 mg/L) was studied, maximum decolorization was observed with 15% inoculum concentration. A significant increase in the activities of laccase (184%) and aminopyrine N-demethylase (300%) in cells obtained after decolorization indicated the involvement of these enzymes in decolorization process. The intermediates formed during the degradation of Crystal Violet were analyzed by gas chromatography and mass spectroscopy (GC/MS). It was detected the presence of N,N,N',N"-tetramethylpararosaniline, [N, N-dimethylaminophenyl] [N-methylaminophenyl] benzophenone, N, N-dimethylaminobenzaldehyde, 4-methyl amino phenol and phenol. We proposed the hypothetical metabolic pathway of Crystal Violet biodegradation by A. radiobacter. Phytotoxicity and microbial toxicity study showed that Crystal Violet biodegradation metabolites were less toxic to bacteria (A. radiobacter, P. aurugenosa and A. vinelandii) contributing to soil fertility and for four kinds of plants (Sorghum bicolor Vigna radiata, Lens culinaris and Triticum aestivum) which are most sensitive, fast growing and commonly used in Indian agriculture.

The decolorization and biodegradation of Reactive Blue 13 (RB13), a sulphonated reactive azo dye, was achieved under static anoxic condition with a bacterial strain identified as Proteus mirabilis LAG, which was isolated from a municipal dump site soil near Lagos, Nigeria. This strain decolorized RB13 (100mg/l) within 5h. The formation of aromatic amine prior to mineralization was supported by Fourier transform infrared spectrometry (FTIR), which revealed the disappearance of certain peaks, particularly those of the aromatic C-H bending at 600-800 cm(-1). Gas chromatography-mass spectrophotometry (GCMS) analysis of the dye metabolite showed the presence of sodium-2(2-formyl-2-hydroxyvinyl) benzoate, with a tropylium cation as its base peak, this suggested the breakage of naphthalene rings in RB13. The detection of azoreductase and laccase activities suggested the enzymatic reduction of azo bonds prior to mineralization. In addition, phytotoxicity studies indicated the detoxification of RB13 to non-toxic degradation products by this strain of P. mirabilis LAG., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Kocuria rosea (MTCC 1532) showed 100% decolorization of methyl orange (50 mg l(-1)) under static condition. The optimum pH and temperature for dye decolorization was 6.8 and 30 degrees C, respectively. The K. rosea (MTCC 1532) showed maximum decolorization of methyl orange when growth medium containing yeast extract as compared to other substrates. The culture exhibited significant ability to decolorize repeated additions of dye, with reduction in time up to 12 h at eighth dye aliquot addition. Significant induction of reductases (NADH-DCIP reductase and azoreductase) suggests its involvement in decolorization of methyl orange. The metabolites formed after decolorization of methyl orange, such as 4-amino sulfonic acid and N,N'-dimethyl p-phenyldiamine were characterized using FTIR and MS. Phytotoxicity and microbial toxicity study showed the methyl orange was toxic and metabolites obtained after its decolorization was nontoxic for experimental plants (Triticum aestivum and Phaseolus mungo) and bacteria (K. rosea, Pseudomonas aurugenosa and Azatobacter vinelandii)., (2009 Elsevier B.V. All rights reserved.)

A microbial consortium DAS consisting three bacterial sp. originally obtained from dye contaminated sites of Solapur, India was selected because it was capable of decolorizing textile effluent and dye faster than the individual bacteria under static conditions. Identification of the isolates by 16S rRNA techniques revealed the isolates to be Pseudomonas species. The concerted metabolic activity of these isolates led to complete decolorization of textile effluent as well as Reactive Orange 16 (100 mg l(-1)) within 48-h at pH 7 and 30 degrees C. Studies involving Reactive Orange 16 (RO16) dye were carried with the bacterial consortium DAS to elucidate the mechanism of biodegradation. Induction of the laccase and reductase enzyme during RO16 decolorization indicated their role in biodegradation. The biodegradation of RO16 was monitored by using IR spectroscopy, HPLC and GC-MS analysis. Cytotoxicity, genotoxicity and phytotoxicity studies carried out before and after decolorization of the textile effluent revealed the nontoxic nature of the biotreated sample.

Micrococcus glutamicus NCIM-2168 exhibited complete decolorization and degradation of C.I. Reactive Green 19A (an initial concentration of 50 mg l(-1)) within 42 h at temperature 37 degrees C and pH 8, under static condition. Extent of mineralization was determined with total organic carbon (TOC) and chemical oxygen demand (COD) measurement, showing a satisfactory reduction of TOC (72%) and COD (66%) within 42 h. Enzyme studies shows involvement of oxidoreductive enzymes in decolorization/degradation process. Analytical studies of the extracted metabolites confirmed the significant degradation of Reactive Green 19A into various metabolites. The microbial toxicity and phytotoxicity assay revealed that the degradation of Reactive Green 19A produced nontoxic metabolites. In addition, the M. glutamicus strain was applied to decolorize a mixture of ten reactive dyes showing a 63% decolorization (in terms of decrease in ADMI value) within 72 h, along with 48% and 42% reduction in TOC and COD under static condition.

Navy blue HER was decolorized and degraded within 24h by Trichosporon beigelii NCIM-3326 under static condition. In the present study, we investigated various physicochemical parameters such as agitation, temperature, pH, cell concentration, initial dye concentration and different carbon and nitrogen sources to achieve maximum dye degradation by T. beigelii. Sequentially, decolorization and decrease in the total organic carbon (TOC) of Navy blue HER by T. beigelii were measured. Among five strains T. beigelii gave the better performance on the decolorization of Navy blue HER along with a 95% TOC reduction within 24h. A significant increase in the activities of NADH-DCIP (dichlorophenolindophenol) reductase and azoreductase in the cells obtained after complete decolorization presumably indicates involvement of these enzymes in decolorization process. UV-vis, TLC, HPLC and FTIR analysis of extracted products confirmed the biodegradation of Navy blue HER. Phytotoxicity study demonstrated no toxicity of the biodegraded products with respect to plants viz. Phaseolus mungo and Sorghum vulgare. In addition to Navy blue HER, this strain also shows ability to decolorize various industrial dyes, including Red HE7B, Golden yellow 4BD, Green HE4BD, Orange HE2R, Malachite green, Crystal violet and Methyl violet.

A developed consortium-GR, consisting of Proteus vulgaris NCIM-2027 (PV) and Micrococcus glutamicus NCIM-2168 (MG), completely decolorized an azo dye Scarlet R under static anoxic condition with an average decolorization rate of 16,666 microg h(-1); which is much faster than that of the pure cultures (PV, 3571 microg h(-1); MG, 2500 microg h(-1)). Consortium-GR gave best decolorization performance with nearly complete mineralization of Scarlet R (over 90% TOC and COD reduction) within 3h, much shorter relative to the individual strains. Induction in the riboflavin reductase and NADH-DCIP reductase was observed in the consortium, suggesting the involvement of these enzymes during the fast decolorization process. The FTIR and GC-MS analysis showed that 1,4-benzenediamine was formed during decolorization/degradation of Scarlet R by consortium-GR. Phytotoxicity studies revealed no toxicity of the biodegraded products of Scarlet R by consortium-GR. In addition, consortium-GR applied for mixture of industrial dyes showed 88% decolorization under static condition with significant reduction in TOC (62%) and COD (68%) within 72 h, suggesting potential application of this microbial consortium in bioremediation of dye-containing wastewater.

Galactomyces geotrichum MTCC 1360 degraded the Scarlet RR (100 mg/l) dye within 18 h, under shaking conditions (150 rpm) in malt yeast medium. The optimum pH and the temperature for decolorization were pH 12 and 50 degrees , respectively. Enzymatic studies revealed an induction of the enzymes, including flavin reductase during the initial stage and lignin peroxidase after complete decolorization of the dye. Decolorization of the dye was induced by the addition of CaCO3 to the medium. EDTA had an inhibitory effect on the dye decolorization along with the laccase activity. The metabolites formed after complete decolorization were analyzed by UV-VIS, HPLC, and FTIR. The GC/MS identification of 3 H quinazolin-4- one, 2-ethylamino-acetamide, 1-chloro-4-nitro-benzene, N- (4-chloro-phenyl)-hydroxylamine, and 4-chloro-pheny-lamine as the final metabolites corroborated with the degradation pathway is suggested to understand the mechanism used by G.geotrichum and thereby aiding development of technologies for the application of this organism to the cleaning-up of aquatic and terrestrial environments.

A novel bacterial strain capable of decolorizing reactive textile dye Red BLI is isolated from the soil sample collected from contaminated sites of textile industry from Solapur, India. The bacterial isolate was identified as Pseudomonas sp. SUK1 on the basis of 16S rDNA analysis. The Pseudomonas sp. SUK1 decolorized Red BLI (50 mg l(-1)) 99.28% within 1h under static anoxic condition at pH range from 6.5 to 7.0 and 30 degrees C. This strain has ability to decolorize various reactive textile dyes. UV-Vis spectroscopy, FTIR and TLC analysis of samples before and after dye decolorization in culture medium confirmed decolorization of Red BLI. A significant increase in the activities of aminopyrine N-demethylase and NADH-DCIP reductase in cells obtained after decolorization indicates involvement of these enzymes in the decolorization process. Phytotoxicity testing with the seeds of Sorghum vulgare and Phaseolus mungo, showed more sensitivity towards the dye, while the products obtained after dye decolorization does not have any inhibitory effects.

The present study dealt with the decolorization and degradation of textile dye Reactive blue-25 (0.1gl(-1)) by mycelium of Aspergillus ochraceus NCIM-1146. Spectrophotometric and visual examinations showed that the decolorization was through fungal adsorption, followed by degradation. Shaking condition was found to be better for complete and faster adsorption (7h) and decolorization (20 days) of dye Reactive blue-25 (100mgl(-1)) as compared to static condition. Presence of glucose in medium showed faster adsorption (4h) and decolorization of dye from bound (7 days) mycelium. FTIR and GCMS analysis study revealed biodegradation of Reactive blue-25 into two metabolites phthalimide and di-isobutyl phthalate.

Saccharomyces cerevisiae MTCC 463 decolourizes toxic azo dye, methyl red by degradation process. Methyl red (100mgl(-1)) is degraded completely within 16min in plain distilled water under static anoxic condition, at the room temperature. Effect of physicochemical parameters (pH of medium, composition of medium, concentration of cells, concentration of dye, temperature and agitation) on methyl red decolourization focused the optimal condition required for decolourization. Biodegradation (fate of metabolism) of methyl red in plain distilled water was found to be pH dependent. Cells of Saccharomyces cerevisiae could degrade methyl red efficiently up to 10 cycles in plain distilled water. Analysis of samples extracted with ethyl acetate from decolourized culture flasks in plain distilled water (pH 6.5) and at pH 9 using UV-VIS, TLC, HPLC and FTIR confirm biodegradation of methyl red into several different metabolites. A study of the enzymes responsible for the biodegradation of methyl red in the control and cells obtained after decolourization in plain distilled water (pH 6.5) and at pH 9 showed different levels of the activities of laccase, lignin peroxidase, NADH-DCIP reductase, azoreductase, tyrosinase and aminopyrine N-demethylase. A significant increase in the activities of lignin peroxidase and NADH-DCIP reductase was observed in the cells obtained after decolourization in plain distilled water (pH 6.5), however cells obtained at pH 9 shows increased activities of azoreductase, tyrosinase, lignin peroxidase and NADH-DCIP reductase. High efficiency to decolourize methyl red in plain distilled water and low requirement of environmental conditions enables this yeast to be used in biological treatment of industrial effluent containing azo dye, methyl red.

Pseudomonas desmolyticum NCIM 2112 was able to degrade a diazo dye Direct Blue-6 (100 mg l(-1)) completely within 72 h of incubation with 88.95% reduction in COD in static anoxic condition. Induction in the activity of oxidative enzymes (LiP, laccase) and tyrosinase while decolorization in the batch culture represents their role in degradation. Dye also induced the activity of aminopyrine N-demethylase, one of the enzyme of mixed function oxidase system. The biodegradation was monitored by UV-Vis, IR spectroscopy and HPLC. The final products, 4-amino naphthalene and amino naphthalene sulfonic acid were characterized by GC-mass spectroscopy.

Administration of 1.5 mg aflatoxin B1 (AFB1)/kg in DMSO as a single i.p. dose to 80 mg phenobarbital/kg i.p. for 3 d pretreated chickens showed significant decrease in the levels of electron transport components and drug metabolizing enzymes compared to phenobarbital administration alone. Induction of mixed function oxidase enzymes due to phenobarbital was not affected by the AFB1 pretreatment. AFB1 administration to 20 mg benzo(a)pyrene/kg in safflower seed oil i.p. for 2 d pretreated chickens showed significant decrease in cytochrome b5 and drug metabolizing enzymes when compared with benzo(a)pyrene treatment alone. However, cytochrome P450 level was slightly higher due to administration of AFB1 to benzo(a)pyrene-injected chickens. The results indicate that the phenobarbital-induced cytochrome P450 is more susceptible to AFB1 than benzo(a)pyrene-induced cytochrome P450.

Administration of Aflatoxin B1 (AFB1) with safflower seed oil to hubbard chickens caused a significant increase in liver microsomal protein, electron transport components, and drug metabolizing enzymes. No alteration was observed in the activity of alanine and aspartate aminotransferases. AFB1 treatment with dimethylsulfoxide (DMSO) as a vehicle caused a significant decrease in electron transport components, drug metabolizing enzymes, and a significant increase in the activity of aspartate aminotransferase. Higher inhibition was observed at 1.5 mg/kg dose level of AFB1. Inhibition by AFB1 was maximal after 24 hr of treatment and decreased thereafter. AFB1 treatment with DMSO caused no significant change in electron transport components and drug metabolizing enzymes in Rhode Red Island (RRI) strain. Vancob male chickens showed significant decrease in electron transport components and drug metabolizing enzymes, while a significant increase was observed in vancob females. Results suggest that the effects of AFB1 depend on treatment vehicle, strain and sex of chickens.

Sulfamethazine (SMZ) administration (ip, 3 d) of different doses to adult male rats showed significant increases in the electron transport components and activities of aminopyrine N-demethylase and aniline hydroxylase at the the 150 mg SMZ/kg dose level. However, 300 mg SMZ/kg doses produced a significant decrease in cytochrome P-450 and the activity of aminopyrine N-demethylase. In a longer duration study, 300 mg SMZ/kg caused significant decreases in cytochrome P-450 and in the activity of aminopyrine N-demethylase. The inducer dose of 150 mg SMZ/kg and the inhibitory dose of 300 mg SMZ/kg were selected for dosing young male, old male and adult female rats. Sulfamethazine administration to young male rats resulted in a significant induction of electron transport components and drug metabolizing enzymes at both dose levels. However, SMZ treatment of old rats produced significant decreases in electron transport components and aminopryine N-demethylase activity at both dose levels. A significant induction in the levels of electron transport components was observed with 150 mg SMZ/kg in female rats. All other parameters were unchanged. Sulfamethazine resulted in a mixed type of inhibition (Ki = 3.5 mM) of aminopyrine N-demethylase in vitro. Hydroxylated metabolites destructed the spectral and catalytic activity of cytochrome P-450. Our studies suggest that SMZ is a substrate of the mixed function oxidase system and induction is dependent on dosage, age and sex of the animals.

Sulfamethazine (SMZ) ip administration for 3 d to chickens showed significant induction of cytochrome P-450 levels and in the activities of aminopyrine N-demethylase, aniline hydroxylase and glutathione s-transferase at the dosage of 150 mg SMZ/kg body weight. Cytochrome P-450 and the activity of aniline hydroxylase were significantly decreased at 300 mg SMZ/kg body weight when compared to 150 mg SMZ/kg body weight. Other doses produced no significant changes in the parameters studied. One hundred fifty mg SMZ/kg for 1 or 3 d caused significant induction of cytochrome b5, cytochrome P-450, the drug metabolizing enzymes and glutathione s-transferase; however, 5 d of SMZ produced no significant changes. The in vitro 10 mM SMZ inhibition of aminopyrine N-demethylase and aniline hydroxylase and the induction of enzymes at 150 mg SMZ/kg body weight indicates that SMZ is a substrate of the mixed function oxidase system and may be an inducer of specific forms of cytochrome P-450.

Dimethyl sulfoxide (DMSO) at a single dose of 3 ml/kg body wt, administered i.p. to male rats, caused a significant increase in the hepatic microsomal aniline hydroxylase activity. However, the level of cytochrome P-450, the activities of NADPH-cytochrome c reductase, benzphetamine N-demethylase and aminopyrine N-demethylase were unchanged at 24 h post-treatment. DMSO interacted with control rat liver microsomes in vitro and produced a type II spectral change (peak at 420 nm and trough at 392 nm). On the other hand, liver microsomes from DMSO-treated rats gave qualitatively similar spectra, but with a higher magnitude of binding. Liver microsomes from DMSO-treated rats showed a 3.4-fold increase in Vmax for aniline hydroxylase, while Km was found to be the same when compared with control rat liver microsomes. In vitro addition of 6 mM DMSO to microsomal incubations from control and DMSO-treated rats caused a 9-fold and a 25-fold increase in Km, respectively, while Vmax values for aniline hydroxylase were unchanged. When DMSO (6 mM) was incubated with rat liver microsomes in the presence of NADPH, there was formation of formaldehyde. The results suggest an interaction of DMSO with microsomal cytochrome P-450.

Intraperitoneal administration of acorn extract of dosage levels of 200, 400 and 600 mg/kg body weight did not produce significant change in the hepatic microsomal cytochrome P-450 levels and the activities of NADPH-cytochrome c reductase, benzphetamine N-demethylase and aniline hydroxylase in young, adult rats (weighing 200-250 g), with the exception of the activity of benzphetamine N-demethylase at the 600 mg/kg dose which was decreased significantly. On the other hand, a dose of only 100 mg/kg body weight ip to old rats (weighing 400-450 g) caused significant decreases in the microsomal cytochrome P-450, benzphetamine N-demethylase and NADPH-cytochrome c reductase activities. However, there was no significant change in the activity of aniline hydroxylase in these rats, indicating selective inhibition of the microsomal enzymes and higher susceptibility of old rats than young ones to acorn toxicants. When the serum samples from the treated young rats were analyzed for sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities as markers of liver toxicity, these activities were significantly higher in the treated rats than the corresponding control values. Similar changes were noted for old rats receiving a dose of 100 mg/kg body weight of acorn extract. The results indicate that acorn extract affects old rats more than young rats as measured by its effect on liver and liver microsomal enzymes.

Simultaneous administration of caffeine (100 mg/kg, i.p., 3 days) and phenobarbital (80 mg/kg, i.p., 3 days) to adult male rats resulted in a significant decrease in hepatic cytochrome P-450 and acetanilide hydroxylase activity, compared to phenobarbital administration alone. While simultaneous administration of caffeine and benzo[a]pyrene (20 mg/kg, i.p., 2 days) increased acetanilide hydroxylase, compared to benzo[a]pyrene administration, no change was seen in the cytochrome P-450 concentration. In vitro addition of 2.5 mM caffeine to microsomal incubations from untreated, phenobarbital- and benzo[a]pyrene-treated rats inhibited aminopyrine N-demethylase activity. No significant difference was seen in the extent of aminopyrine N-demethylase inhibition due to the in vitro addition of caffeine to microsomes from untreated or phenobarbital-treated rats, whereas inhibition in microsomes from benzo[a]pyrene-treated rats was greater.

Administration of caffeine, ip 100 mg/kg/day for 1-5 days, to adult male rats resulted in a significant increase in hepatic cytochrome P-450 and b5 concentrations and in cytochrome c reductase, aminopyrine N-demethylase and acetanilide hydroxylase activities. No change was seen in relative liver weight but microsomal protein content was increased after treatment for 1 day and decreased after treatment for 3 or 5 days. In adult rats given 25, 100 or 150 mg caffeine/kg for 3 days, maximum stimulation of mixed-function oxidases was seen with the 100-mg/kg dose. Caffeine treatment (100 mg/kg for 3 days) increased relative liver weight in female guinea-pigs and decreased it in chicks and female mice, and decreased microsomal protein content in male mice, female guinea-pigs and young rats, and increased it in chicks. A significant increase in hepatic cytochrome P-450 content was seen in all species studied. Cytochrome b5 content was increased in chicks and young rats, while cytochrome c reductase activity was increased in male and female mice, young rats and chicks and decreased in female guinea-pigs. Aminopyrine N-demethylase activity was increased in young rats and female guinea-pigs, and acetanilide hydroxylase was increased in all test species except male mice. In vitro addition of 2.5 mM-caffeine to microsomal incubations from untreated rats, guinea-pigs, mice and chicks inhibited aminopyrine N-demethylase activity, although only to a significant extent in male mice; addition of caffeine to incubations containing microsomes from caffeine-treated animals produced significant inhibition of aminopyrine N-demethylase activity in microsomes from adult and young rats and female guinea-pigs. Aminopyrine N-demethylase inhibition did not increase with increasing concentration of added caffeine, although acetanilide hydroxylase activity was progressively inhibited in the microsomal incubates from both control and caffeine-treated animals.

Cytochrome P-450 substrate interactions were studied with cytochrome P-450 partially purified from livers of untreated, phenobarbital-treated, benzo[a]pyrene-treated and caffeine-treated rats. Partial inhibition of aminopyrine N-demethylase in presence of in vitro caffeine observed with intact microsomes was further investigated in a reconstituted system composed of partially purified cytochrome P-450 and cytochrome c reductase. Caffeine addition (in vitro) to partially purified cytochrome P-450 altered the hexobarbital, aniline and ethylisocyanide induced spectral change, and decreased NADPH oxidation in presence of substrates aminopyrine and acetanilide. NADPH oxidation was found to be increased in presence of aminopyrine and unaltered in presence of acetanilide in reconstituted system having partially purified cytochrome P-450 from caffeine-treated rats. Our studies suggest that caffeine acts as a true modifier of cytochrome P-450 and is possibly responsible for the formation of abortive complexes with aminopyrine.

The effect of ampicillin [(D)-alpha-aminobenzyl penicillin] administration on the hepatic mixed-function oxidase (MFO) system was studied in male mice. Ampicillin (100 mg/kg, i.p., 3 days) decreased the levels of cytochrome P-450, aminopyrine N-demethylase, acetanilide hydroxylase and cytochrome c-reductase activity significantly. In carbon tetrachloride (CCl4)-pretreated mice, ampicillin increased acetanilide hydroxylation compared with CCl4 treatment alone; however, all other parameters of the MFO system remained unchanged. Ampicillin exhibited type II binding with microsomes (trough at 388 nm, peak at 430 nm). Thus ampicillin acts as an inhibitor of the MFO system.