A novel tumor necrosis factor-alpha inhibitory protein, TIP-B1.

TIP-B1, a novel TNF inhibitory protein, has been identified, purified and characterized from cytosolic extracts of TNF-treated human fibroblasts, and a partial TIP-B1 cDNA clone has been obtained. The (27 kDa pI approximately 4.5 TIP-B1 protein is unique based on both the sequence of three internal peptides (comprising 51 amino acids), and the nucleotide sequence of the corresponding cDNA clone. TNF-sensitive cells, when exposed to TIP-B1 prior to the addition of TNF, are completely protected from TNF-induced lysis. Thus, this TIP-B1 treatment effectively makes these cells TNF-resistant. Furthermore, TIP-B1 protects cells from apoptotic lysis induced by TNF. TIP-B1 does not interfere with the interactions between TNF and the TNF receptors based on flow cytometric analysis of the cellular binding of biotinylated TNF. These and other data indicate that TIP-B1 is not a soluble TNF receptor, nor an anti-TNF antibody, nor a protease that degrades TNF, yet TIP-B1 functions when added exogenously to cells. Thus, TIP-B1 is not one of the proteins previously reported to be involved in resistance to TNF. The fact that incubation of the newly discovered novel TIP-B1 with TNF-sensitive cells protects them from TNF-induced cell death, including TNF-mediated apoptosis, makes TIP-B1 a candidate for therapeutic modulation of TNF-induced effects.

Identification, characterization, and cloning of TIP-B1, a novel protein inhibitor of tumor necrosis factor-induced lysis.

Some cancer cells evade elimination by virtue of their insensitivity to agents that induce apoptosis. Conversely, the side effects of anticancer agents could be diminished if normal cells were more resistant. To further elucidate the factors that contribute to the susceptibility of a cell to apoptosis, these investigations were designed to identify proteins isolated from cells exposed to low concentrations of tumor necrosis factor (TNF) that, when incubated with normally TNF-sensitive cells, protect these cells from TNF-induced cytotoxicity. TIP-B1, a novel protein, has been identified, purified, and characterized from cytosolic extracts of TNF-treated human fibroblasts. The approximately 27 kDa pI-4.5 TIP-B1 protein is unique based on both the sequence of three internal peptides (comprising 51 amino acids) and the nucleotide sequence of the corresponding 783-bp cDNA partial clone. Western blot analyses using polyclonal antisera raised against both the purified native TIP-B1 and the approximately 14 kDa product of the cDNA partial TIP-B1 clone, as well as Northern blot analyses using the cDNA insert as a probe, indicate that TIP-B1 may belong to a family of proteins that are expressed in a number of cell lines from diverse tissues. TNF-sensitive cells, when exposed to 4-10 microg/ml concentrations of TIP-B1 prior to the addition of TNF, are completely protected from TNF-induced lysis. Furthermore, TIP-B1 protects cells from apoptotic lysis induced by TNF. Preincubation of TIP-B1 with TNF does not affect the ability of TNF to induce lysis. Moreover, TIP-B1 does not seem to interfere with the interactions between TNF and the TNF receptors, based on a preliminary flow cytometric analysis of the cellular binding of biotinylated TNF. On the basis of these characteristics, TIP-B1 is not a soluble TNF receptor, an anti-TNF antibody, nor a protease that degrades TNF; yet TIP-B1 functions when added exogenously to cells. These characteristics, its novel sequence, and its function when added exogenously to cells indicate that TIP-B1 is unique and is not one of the other proteins reported previously to be involved in resistance to TNF. The ability of TIP-B1 to function after exogenous incubation with target cells makes TIP-B1 a likely candidate for therapeutic manipulation of TNF-induced effects.

A novel TNF-inducible message with putative growth suppressor function.

We report the nucleotide sequence of a novel cDNA and TNF-induced expression of the corresponding message (mRNA) in human fibroblast cells. This message is also expressed in certain human tumor cell lines and is over-expressed in a colon cancer cell line (HT-29). NIH3T3 cells transfected with the antisense construct of the 5'-region of this novel cDNA formed 20-fold more colonies in culture compared to cells transfected with a sense construct of the same region or the sense and the antisense constructs of the central region of this cDNA. This observation suggests a possible growth suppressor function for the gene represented by this cDNA.

Mutations induced by monofunctional and bifunctional phosphoramide mustards in supF tRNA gene.

The relative mutagenicity, nature of the mutations and the sequence specificity of mutations induced by the bifunctional alkylating agent, phosphoramide mustard (PM) and a monofunctional derivative, dechloroethyl phosphoramide mustard (dePM), were analyzed by the Ames test and by an in vitro shuttle vector mutagenesis assay. Both PM and dePM increased the mutation frequency above background in either assay. However, on an equimolar basis, dePM was less mutagenic than PM. In the in vitro shuttle vector mutagenesis assay, sequencing demonstrated that about 40% of the mutant plasmids contained more than one mutation in the supF tRNA gene segment of the plasmid. About 70% of the mutations observed in dePM-treated plasmids were single base substitutions with A:T and G:C base pairs being mutated at equivalent rates. In contrast, only about 50% of the mutations observed in PM-treated plasmids were single base substitutions, 80% of which involved G:C base pairs. Single base deletions and insertions were found in approximately equal proportions with both compounds; however, these lesions were in greater abundance in PM-treated plasmids. Putative hot-spots for mutation in the supF tRNA gene included base pairs at position 102 and 110 for PM and positions 170 and 171 for dePM.

Mutations induced in a shuttle vector plasmid exposed to monofunctionally activated mitomycin C.

Reductive activation of mitomycin C leads to its covalent binding to DNA, forming monoadducts and cross-links. The cytotoxicity of mitomycin C has been attributed to cross-link formation, whereas monoadducts are assumed to cause mutagenicity. We have developed a 32P-postlabeling technique to measure mitomycin C DNA adducts. Using this technique, we have measured monoadduct formation in the shuttle vector plasmid pSP189 and have determined mutations induced by monoadduct formation. The shuttle vector plasmid was incubated with mitomycin C under conditions favoring monofunctional activation of mitomycin C. The plasmid was then replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence of pSP189. One major mitomycin C/DNA adduct was observed by 32P-postlabeling and was characterized as a monoadduct of guanine. When pSP189 was exposed to monofunctionally activated mitomycin C, increases in adduct levels and mutation frequency were found to be related to mitomycin C concentration. The majority of the mutations involved single bases, with base substitutions making up 59.1% of the total mutations observed. Of the base substitutions, 67.2% were transversions and 32.8% were transitions, with nearly 80% of all base substitutions involving G:C base pairs. Deletions, either as single bases or large deletions, also involved G:C base pairs the majority of the time. The observed bias of mutations at G:C and the formation of a mitomycin C/DNA monoadduct involving guanine suggests that monoadduct formation may be responsible for the mutations.

Mutations induced by dacarbazine activated with cytochrome P-450.

The mutagenicity of the antitumor drug dacarbazine (DTIC) is due to alkylation of cellular DNA by metabolites resulting from the metabolism of this drug by the mixed function oxidase system. In the present study, we used an in vitro shuttle vector assay to study the base and sequence specificity of mutagenesis by DTIC. The shuttle vector plasmid pSP189 was treated with DTIC (1-2.5 mM) in vitro in a reconstituted cytochrome P-450 system at 37 degrees C for either 30 or 60 min. SupF tRNA gene insert contained in the plasmid was sequenced after replication of the drug-treated plasmid in human Ad 293 cells followed by amplification in indicator bacteria. Mutagenesis of DTIC in this system was dependent upon the presence of the cytochrome P-450 reconstituted system and NADPH. Mutations induced by DTIC included single base substitutions (35%), single base deletions (30.5%), single base insertions (19.4%) and large deletions (13.8%). Among the substitutions, transversions and transitions were in the ratio of 1:0.7. Base pairs 108 and 127 in the SupF tRNA of the pSP189 were identified as mutational hot spots.

Formation of 8-hydroxyguanine in DNA during mitomycin C activation.

DNA damage caused indirectly via reactive oxygen species generated during reductive activation of mitomycin C was evaluated. This oxidative DNA damage was measured by determining the formation of 8-hydroxyguanine in DNA exposed to chemically or enzymatically activated mitomycin C. The level of 8-hydroxyguanine was measured indirectly by determining formamidopyrimidine-DNA glycosylase-sensitive sites induced in plasmid DNA exposed to mitomycin C and directly by a 32P-postlabeling assay for the modified base. Activation of mitomycin C by sodium borohydride in air, by H2/Pt, or xanthine oxidase in N2 caused increases in the level of 8-hydroxyguanine. The extent of the increase varied according to the incubation conditions with the greatest increase being observed in DNA exposed to mitomycin C activated under hypoxic conditions. These results support a possible indirect mechanism for DNA damage caused by mitomycin C that is mediated by reactive oxygen species.

Mutations in a shuttle vector exposed to activated mitomycin C.

The cytotoxicity of the potent antibiotic and antitumor agent mitomycin C (MMC) is due to its irreversible binding to DNA. Alkylating species generated by bioreductive activation of MMC are known to cause monoadducts and cross-links in DNA by specifically binding to guanine residues. To gain insight into how these lesions lead to base- and sequence-specific mutations, shuttle vector pSP189 was treated with MMC chemically reduced by treatment with sodium borohydride, replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence. The MMC-induced mutations were predominantly base substitutions. Eighty-four percent of the base substitutions were transversions, with G:C-->T:A the major transversion. Single base deletions were the other major mutational event, and 77% of these were G:C deletions. Base positions 115, 123, and 163 were mutational hot spots based on the frequency of independent mutations. Identification of a single MMC adduct (presumed to be a modified G on the basis of its Rf value) and clustering of MMC-induced mutations at three GC-rich areas (nt 100-123, 152-163, and 168-176) suggested that the mutational spectrum we found was due to binding of MMC to guanine on either strand of the plasmid DNA.

Altered expression of cytochrome P450 mRNA during chemical-induced hepatocarcinogenesis and following partial hepatectomy.

Levels of various cytochrome P450 proteins have been reported to be decreased to varying degrees in chemically induced hepatocyte nodules and following partial hepatectomy (PH). By screening a rat liver lambda ZAP cDNA expression library with antibodies raised against a partially purified preparation of cytochrome P450 isolated from untreated male Fischer 344 rats, we have isolated a 1.1-kb cDNA. This cDNA was sequenced for 139 bases from the 5' end of the sense strand and comparison of the resulting sequence with the sequences in Gene Man DNA data bank revealed 95% homology of the sequenced portion with male-specific rat cytochrome P450 (M-1, CYP IIC11). The 32P-labeled cDNA was used as a hybridization probe on RNA blots (Northern blots) prepared with total RNA from rat livers obtained post PH, from aflatoxin B1(AFB1)-induced rat liver tumors and from rat liver nodules induced with a combination of diethylnitrosamine/acetylaminofluorene/PH (DEN/AFF/PH). At 36 and 72 hr post PH, the mRNA level was decreased by > 93%. Relative to the corresponding control livers, the mRNA level was also decreased by 97% in the liver nodules and by 57% in AFB1-induced liver tumors. The RNA blots derived from the liver nodules and AFB1-induced liver tumors were also probed with a cDNA probe (R17) that recognizes other cytochromes P450 (CYP IIB1/CYP IIB2). The mRNA corresponding to CYP IIB1/CYP IIB2 was also depressed 92% in the nodules and 65% in the tumors. These results clearly indicate that the depression of both CYP IIC11 and IIB1/IIB2 in the hepatic nodules and the tumors is related to the inhibition of transcription and/or enhanced degradation of the mRNA.

32P-postlabeling of acrolein-deoxyguanosine adducts in DNA after nuclease P1 digestion.

In order to study the relationship between the level of acrolein-DNA adducts and their biological effects, sensitive methods are needed to quantitate DNA adducts. 32P-postlabeling is one such method that has been widely used and we have adapted the technique to detect acrolein-deoxyguanosine adducts. Adducts formed by the reaction of acrolein and deoxyguanosine-3'-monophosphate were isolated by HPLC. Based on their UV spectra and cochromatography with standards after dephosphorylation with acid phosphatase, these adducts were identified as the nucleotide equivalents of cyclic 1,N2-propanodeoxyguanosine adducts formed by acrolein that have been described by Chung et al. [15]. As nucleotides, the adducts were good substrates for polynucleotide kinase-mediated transfer of phosphate from ATP and were able to be detected by 32P-postlabeling. These adducts were resistant to the activity of nuclease P1 and dinucleoside monophosphates in the form d(G*pN) where G* is the acrolein-guanine adduct also resisted digestion by nuclease P1. Digestion of DNA by nuclease P1 and acid phosphatase resulted in the conversion of normal nucleotides to nucleosides and selective enrichment of the adducts as dinucleoside monophosphates. Using nuclease P1/acid phosphatase digestion, followed by 32P-postlabeling and TLC separation, levels of the two adducts in acrolein-treated DNA were found to be about 6185 and 19,222 nmol/mol.

Aflatoxin-transformed C3H/10T1/2 cells overexpress protein kinase C and have an altered response to phorbol ester treatments.

The aflatoxin B1-transformed C3H/10T1/2 (10T1/2) cell line 7SA has disordered growth in culture and is tumorigenic in syngeneic mice. Chronic exposure (14 days) of 10T1/2 and 7SA cells to phorbol 12,13-dibutyrate (PDBu) increased the saturation density of 10T1/2 cells but dramatically slowed the entry of 7SA cells into the log phase of growth without affecting their final saturation density. Similar PDBu treatment of low-density cultures dramatically decreased the size of 7SA colonies. Both cell lines bound [3H]PDBu in a specific and saturable manner. Analysis of this binding yielded linear Scatchard plots for both cell lines with distinctly different Kd values (10.7 nM for 10T1/2 versus 54.5 nM for 7SA). The total amount of [3H]PDBu bound was 2-fold greater in the 7SA cells versus the 10T1/2 cell line. Both cell lines released arachidonic acid following a 2-h exposure to PDBu; however, the magnitude of the response of the 7SA cells was only one-half that of the 10T1/2 cells. Western blot analysis of protein kinase C (PKC) using specific anti-PKC antibodies revealed a greater total amount of PKC alpha in the 7SA cells relative to an equal number of 10T1/2 cells. No immunoreactive PKC alpha was found in either cell line 16 h after exposure to 600 nM PDBu; however, PKC alpha returned to control levels in both cell lines 24 h after removal of the phorbol ester. These results suggest that an overexpression of PKC alpha may play a role in the altered biological properties of aflatoxin-transformed 10T1/2 cells.

A cyclophosphamide/DNA phosphoester adduct formed in vitro and in vivo.

The antitumor activity of cyclophosphamide is thought to be due to the alkylating activity of phosphoramide mustard, a metabolite of cyclophosphamide. Reaction of 2'-deoxyguanosine 3'-monophosphate and phosphoramide mustard resulted in the formation of several adducts that could be detected by high performance liquid chromatography (HPLC). One of these adducts, isolated and purified by HPLC, could be detected by 32P postlabeling. This product was identified by UV, nuclear magnetic resonance, and mass spectrometry and by acid, base, and enzymatic hydrolysis to be 2'-deoxyguanosine 3'-monophosphate 2-(2-hydroxyethyl)aminoethyl ester. A combination of HPLC fractionation of digested DNA and 32P postlabeling was used to detect this adduct in calf thymus DNA incubated in vitro with metabolically activated cyclophosphamide and in DNA from the liver of mice treated with cyclophosphamide. In these DNA samples the adduct occurred at a level of 1/10(5) and 1/3 x 10(7) nucleotides, respectively.

Altered benzo[a]pyrene metabolism in C3H/10T1/2 cells transformed by aflatoxin B1 or 3-methylcholanthrene.

C3H/10T1/2 clone 8 (10T1/2) cells possess Phase I and Phase II xenobiotic metabolizing enzymes associated with the metabolism of polycyclic aromatic hydrocarbons to activated or detoxified species. We compared the metabolism of benzo[a]pyrene (BaP) by these cells to an aflatoxin B1 (AFB1)-transformed line (7SA) and a 3-methylcholanthrene (3-MC)-transformed line (MCA) isolated from carcinogen-treated 10T1/2 cells. Relative to 10T1/2 cells, basal levels of cytochrome P450-mediated aryl hydrocarbon hydroxylase (AHH) were significantly depressed in 7SA cells by about 30%. The inducibility of AHH by BaP treatment was depressed by 30-70% in MCA and 7SA cells over a 36-hr time course. 10T1/2 and MCA cells accumulated similar intracellular amounts of 3-OH-BaP by 12 and 24 hr, respectively; in contrast the accumulation of 3-OH-BaP in 7SA cells was 70% lower. During 36 hr of BaP treatment, total BaP-DNA adduct levels formed in 7SA and MCA cells, determined by 32P-postlabeling analysis, were 90 and 83% lower, respectively, than those found in 10T1/2 cells. These differences in response to BaP treatment were not related to cellular differences in the uptake or efflux of BaP. Relative to 10T1/2 or MCA cells, 7SA cells were found to have at least a twofold increase in UDP-glucuronyltransferase activity, which correlated with the lower intracellular accumulation of 3-OH-BaP and enhanced formation of extracellular polar metabolites. MCA cells had an almost twofold increase in glutathione S-transferase activity relative to parental 10T1/2 cells but produced lower levels of extracellular polar metabolites. These results demonstrate an association between chemical transformation of 10T1/2 cells and altered xenobiotic metabolism. This system may provide an in vitro model for examining the molecular events responsible for the biochemically altered phenotype of the malignantly transformed cell.

32P-postlabeling analysis of binding of the cyclophosphamide metabolite, acrolein, to DNA.

The cyclophosphamide metabolite, acrolein, was reacted with 2'-deoxyguanosine-3'-monophosphate, and two adducts were detected by high performance liquid chromatography and 32P-postlabeling assay. These adducts were resistant to dephosphorylation by nuclease P1 and could be isolated and detected from calf thymus DNA that had been reacted in vitro with acrolein. A combination of HPLC purification and enzymatic digestion of normal nucleotides by nuclease P1 allowed for the detection of these adducts in hepatic DNA from mice treated with cyclophosphamide. The level of the two adducts in the hepatic DNA, as determined by 32P-postlabeling, was one adduct per 2.7-4.1 x 10(7) normal nucleotides.

Formation of a phosphoramide mustard-nucleotide adduct that is not by alkylation at the N7 position of guanine.

The reaction of 2'-deoxyguanosine 3'-monophosphate with phosphoramide mustard resulted in the formation of several adducts. One of these adducts was formed by linking phosphoramide mustard to the phosphate group of 2'-deoxyguanosine 3'-monophosphate rather than by the generally accepted mechanism involving alkylation at the N7 position of guanine. This adduct served as an acceptor for the transfer of 32p from [gamma 32P]ATP by polynucleotide kinase and thus could be detected by the sensitive 32p-postlabeling assay.

Induction of aryl hydrocarbon hydroxylase and DNA adduct formation in parental and carcinogen transformed C3H/10T1/2 clone 8 cells by benzo[a]pyrene.

C3H/10T1/2 clone 8 (10T1/2) cells possess aryl hydrocarbon hydroxylase (AHH) activity capable of metabolizing polycyclic aromatic hydrocarbons to ultimate carcinogenic forms. AHH activity in 10T1/2 cells was measured before and after culturing in the presence of benzo[a]pyrene (B[a]P), and compared to the AHH activity found in carcinogen-transformed 10T1/2 cell lines treated similarly. The cell lines were also examined for B[a]P-DNA adduct formation, using the 32P-postlabelling technique. Treatment of parental 10T1/2 cells with B[a]P was found to significantly increase AHH activity and produce substantial numbers of DNA adducts. In addition to a major B[a]P-DNA adduct, 5-6 minor DNA adducts were also detected. Relative to parental 10T1/2 cells, an aflatoxin B1-transformed 10T1/2 cell line (7SA) was found to have significantly depressed AHH activity. In addition, after treatment with B[a]P, 7SA cells had only 8% of the B[a]P-DNA adduct levels found in 10T1/2 cells. This system may provide an in vitro model for investigating mechanisms responsible for the depression of cytochrome P-450 activities by chemical carcinogens.

The effect of inducers of mixed-function oxidases on hepatic microsome-mediated aflatoxin B1 transformation in C3H/10T1/2 cells.

The potent hepatotoxin and hepatocarcinogen aflatoxin B1 (AFB1) is metabolized by different forms of cytochrome P450 associated with the hepatic mixed-function oxidase system. C3H/10T1/2(10T1/2) cells, which have limited inherent capacity to metabolize AFB1, were treated with AFB1 in the presence of hepatic microsomes isolated from chemically treated rats to investigate the effects of the induction of specific cytochromes P450 on AFB1-mediated toxicity and transformation. Relative to uninduced microsomes, phenobarbital (PB) treatment induced AFB1-DNA binding (essentially representing the formation of AFB1-8,9-oxide bound to DNA) 3.2-fold, while pretreatment with Aroclor 1254, 3-methylcholanthrene (3-MC), or 5,6-benzoflavone (beta-NF) preferentially induced aflatoxin M1 (AFM1) formation from 2- to 5-fold. 10T1/2 cells were exposed to a multiple treatment regimen with 4 microM AFB1 and hepatic microsomes from uninduced, PB-, Aroclor 1254-, 3-MC-, or beta-NF-treated rats; respective cumulative toxicities of approximately 90, 95, 70, 60, and 40% control (no microsomes) values resulted. An enhanced AFB1 transformation response correlated with the increasing toxicities observed for the different treatments, with uninduced or PB-induced microsomes yielding approximately four foci/dish, while treatments with Aroclor 1254-, 3-MC-, or beta-NF-induced microsomes resulted in only one to two foci/dish. These results demonstrate that AFB1 is a complete carcinogen in the 10T1/2 system when repetitive incubations are used in conjunction with an appropriate hepatic microsomal activation system. These data also correlate the induction of AFB1-4-hydroxylase with a decrease in AFB1-mediated toxicity and transformation of 10T1/2 cells, and support the hypothesis that the Phase I metabolic conversion of AFB1 to AFM1 in the liver represents an effective detoxification pathway per se.

Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase.

Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.

The genetics of aflatoxin B1 metabolism. Association of the induction of aflatoxin B1-4-hydroxylase with the transcriptional activation of cytochrome P3-450 gene.

The association between murine cytochrome P3-450 and hepatic aflatoxin B1-4-hydroxylase, a cytochrome P-450-dependent enzyme which converts aflatoxin B1 (AFB1) to aflatoxin M1 (AFM1), was examined by (a) purification of the cytochrome P-450 which preferentially metabolizes AFB1 to AFM1; (b) isolation of the specific cDNA clone; and (c) correlating induction of transcriptional activation of the specific message with the enzyme activity in the hepatic microsomes. Isolation of cytochromes P-450 from C57BL/6 mice, an Ah-responsive strain, pretreated with a 150 mg/kg dose of beta-naphthoflavone resulted in the partial purification of the cytochrome P-450 with preference for the metabolism of AFB1 to AFM1. Antibodies raised against this cytochrome P-450 were used to enrich hepatic mRNA for cDNA cloning. A cDNA library screened with a rat cytochrome P-450c gene probe yielded only two types of cDNA clones that contained inserts corresponding to cytochrome P1-450 and cytochrome P3-450. Specific restriction fragments of near full-length P1-450 cDNA and full-length P3-450 cDNA, hybridizing only with their respective messages, were isolated and used to assess transcriptional activation of these messages in liver and extrahepatic tissues from C57BL/6 mice treated with 3-methylcholanthrene, beta-naphthoflavone, indolylacetonitrile, and Aroclor-1254. Dose-dependent induction of the two messenger RNAs, when compared with the induction of specific enzyme activities, demonstrated the association of cytochrome P1-450 with aryl hydrocarbon hydroxylase activity and the association of cytochrome P3-450 with AFB1-4-hydroxylase activity. This supports our earlier hypothesis that AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase, although regulated by the Ah locus, are the products of two separate genes (Gurtoo, H.L., Dahms, R.P., Kanter, P., and Vaught, J.B. (1978) J. Biol. Chem. 253, 3952-3961).

Lipid peroxidation induced by cyclophosphamide.

Intraperitoneal administration of a single dose of cyclophosphamide (CP) to rats was found to produce hepatic glutathione depletion and to enhance NADPH-mediated lipid peroxidation in the 15,000 x g supernatant fraction of the liver. These effects were associated with CP in a dose- and a time-dependent manner. The data suggest that the glutathione depletion is, at least in part, responsible for the enhancement in lipid peroxidation induced by CP.