Toxicity of xanthene food dyes by inhibition of human drug-metabolizing enzymes in a noncompetitive manner.

The synthetic food dyes studied were rose bengal (RB), phroxine (PL), amaranth, erythrosine B (ET), allura red, new coccine, acid red (AR), tartrazine, sunset yellow FCF, brilliant blue FCF, and indigo carmine. First, data confirmed that these dyes were not substrates for CYP2A6, UGT1A6, and UGT2B7. ET inhibited UGT1A6 (glucuronidation of p-nitrophenol) and UGT2B7 (glucuronidation of androsterone). We showed the inhibitory effect of xanthene dye on human UGT1A6 activity. Basic ET, PL, and RB in those food dyes strongly inhibited UGT1A6 activity, with IC(50) values = 0.05, 0.04, and 0.015 mM, respectively. Meanwhile, AR of an acidic xanthene food dye showed no inhibition. Next, we studied the inhibition of CYP3A4 of a major phase I drug-metabolizing enzyme and P-glycoprotein of a major transporter by synthetic food dyes. Human CYP3A4 and P-glycoprotein were also inhibited by basic xanthene food dyes. The IC(50) values of these dyes to inhibit CYP3A4 and P-glycoprotein were the same as the inhibition level of UGT1A6 by three halogenated xanthene food dyes (ET, PL, and RB) described above, except AR, like the results with UGT1A6 and UGT2B7. We also confirmed the noninhibition of CYP3A4 and P-gp by other synthetic food dyes. Part of this inhibition depended upon the reaction of (1)O(2) originating on xanthene dyes by light irradiation, because inhibition was prevented by (1)O(2) quenchers. We studied the influence of superoxide dismutase and catalase on this inhibition by dyes and we found prevention of inhibition by superoxide dismutase but not catalase. This result suggests that superoxide anions, originating on dyes by light irradiation, must attack drug-metabolizing enzymes. It is possible that red cosmetics containing phloxine, erythrosine, or rose bengal react with proteins on skin under lighting and may lead to rough skin.

Genuine functions of P-glycoprotein (ABCB1).

P-glycoprotein (P-gp, ABCB1, MDR1) was recognized as a drug-exporting protein from cancer cells three decade ago. Apart from the multidrug transporter side effects of P-gp, normal physiological functions of P-gp have been reported. P-gp could be responsible for translocating platelet-activating factor (PAF) across the plasma membrane and PAF inhibited drug transport mediated by P-gp in cancer cells. P-gp regulated the translocation of sphingomyelin (SM) and GlcCer, and short chain C(6)-NBD-GlcCer was found in the apical medium of P-gp cells exclusively and not in the basolateral membrane. SM plays an important role in the esterification of cholesterol. High expression of P-gp prevents stem-cell differentiation, leading to the proliferation and amplification of this cell repertoire, and functional P-gp plays a fundamental role in regulating programmed cell death, apoptosis. The transporter function of P-gp is therefore necessary to protect cells from death. P-gp can translocate both C(6)-NBD-PC and C(6)-NBD-PE across the apical membrane. This PC translocation was also confirmed with [(3)H]choline radioactivity. Progesterone is not transported by P-gp, but blocks P-gp-mediated efflux of other drugs and P-gp can mediate the transport of a variety of steroids. Cells transfected with human P-gp esterified more cholesterol. P-gp might also be involved in the transport of cytokines, particularly IL-1beta, IL-2, IL-4 and IFNgamma, out of activated normal lymphocytes into the surrounding medium. P-gp expression is also associated with a volume-activated chloride channel, thus P-gp is bifunctional with both transport and channel regulators. We also present information about P-gp polymorphism and new structural concepts, "gate" and "twist", of the P-gp structure.

Study of oxidized lipids as endogenous substrates of P-gp (ABCB1).

We studied endogenous substrates for P-glycoprotein (P-gp) in an oxidative reaction mixture of ceramides, phospholipids, sphingolipids, or GM1-gangliosides (GM1-G). Extracts from the reaction mixture of galactocerebrosides (GalCer), sphingomyelin (SM) , lactocerebrosides (LactoCer), and asolectine (AS) with 0.3% hydrogen peroxide exhibited significant ATPase activity of P-gp of 7.6, 7.8, 5.3, and 4.7 nmol/min/mg protein, respectively, at a concentration of 10 microg equivalent/ml, but not GalCer, SM, LactoCer, and AS themselves. Meanwhile, both GM1-G and its oxidized product showed ATPase activity of 3.7 nmol/min/mg protein at a concentration of 0.75 microM. Phosphatidylcholine, phosphatidylethanolamine, phophatidylserine, triglyceride, and cholesterol did not show P-gp activity. When reactive oxygen species, such as hydrogen peroxide, exceed the ability of antioxidant defense systems to remove it from living cells, SM, GalCer, LactoCer, and AS could react with it; therefore, it is possible for these oxidized lipids to play as substrates for P-gp in living cells. This finding should be a milestone to search a new physiological P-gp function.

Induction of human UGT1A1 by bilirubin through AhR dependent pathway.

UDP-glucuronosyltransferase1A1 (UGT1A1) plays a key role to conjugate bilirubin and preventing jaundice, but there is no report showing the induction of human UGT1A1 (UGT1A1) by bilirubin. In this report, we show findings of the induction of the reporter gene (-3475/+14) of UGT1A1 in HepG2 cells by bilirubin at 50 microM, 100 microM, with human aryl hydrocarbon receptor (hAhR). We confirmed that induction of the reporter gene by bilirubin is dependent on the position of the xenobiotic responsive element (XRE) (-3328/-3319) of UGT1A1, because the XRE deletion UGT1A1 gene did not respond to stimulation by a complex of bilirubin and hAhR. alpha-Naphthoflavone (alpha-NF) of a typical AhR antagonist at 50 microM inhibited induction by bilirubin, suggesting that bilirubin stimulates through binding with hAhR. Meanwhile, bilirubin itself did not stimulate the induction of AhR, because we detected no-elevation of the mRNA level of AhR by RT-PCR. These results indicate that the induction of UGT1A1 by bilirubin-AhR did not depend on the elevation of AhR but on ligand binding. From this result, we considered that high bilirubin in neonates must induce the elevation of UGT1A1 after birth to prevent jaundice, and bilirubin in adults also regulates the level of UGT1A1. This is the first report showing direct induction of UGT1A1 by a bilirubin through AhR pathway.

Induction of human UGT1A1 by a complex of dexamethasone-GR dependent on proximal site and independent of PBREM.

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays a key role to conjugate bilirubin and prevent jaundice. There are two major elements for the induction of UGT1A1, such as PBREM (-3483/-3194), far from the promoter site, and HNF1 (-75/-63), near to the promoter site. In a previous report, we showed that the proximal HNF1 site is essential for the induction of UGT1A1 by glucocorticoid receptor (GR). In this report, we investigated the influence of PBREM on the induction of the UGT1A1 reporter gene by GR and PXR with dexamethasone (DEX). We confirmed that GR was transferred from cytosol into the nucleus in 15-30 min by DEX stimulation, but HNF1 was not. We constructed a reporter gene containing PBREM to compare the induction of the reporter gene without PBREM by DEX-GR. The results show that induction of the reporter gene with PBREM by DEX at 100 muM is the same level as the induction of the reporter gene without PBREM, although PBREM contains GRE. Co-transfection of hGR with the reporter gene did not show any influence of the induction of the reporter gene between the vector with and without PBREM. Meanwhile, by co-transfection of hPXR, the induction of the reporter gene with PBREM was significantly more than the induction of the reporter gene without PBREM at 100 microM DEX. This supports that hPXR induced UGT1A1 through PBREM by DEX. These results showed that PBREM has no relation with the induction by DEX-GR but the proximal site of UGT1A1 may function in stimulation by DEX-GR.

The N-terminal of human UGT1A6 is on the outside, as evidenced by ELISA with autoantibody in autoimmune hepatitis sera.

Sera from AIH (autoimmune hepatitis) type 2 patients contain an autoantibody against the UGT1A subtype, called anti-LKM3. Previously, we reported that sera in AIH type 1 patients contained autoantibodies against drug-metabolizing enzymes (Shinoda et al. (2004) Autoimmunity, 37, 473). In this report, we showed that AIH-1 sera did not react with some peptides in the C-terminal half of the UGT1A subtype but reacted with a peptide P1(33-42) among several common peptides in the N-terminal half of the UGT1A subtype. This result suggests that the P1 site (33-42) presents on the outside of the UGT1A molecule to be recognized by lymphocytes of the immune system to produce an autoantibody. To detect a key recognition site on peptide P1(33-42), we studied the reactivity of two peptides, M1(28-37) and M2(38-47), containing the N-terminal and C-terminal half of peptide P1. Peptide M2 did not react with AIH-1 serum but peptide M1 did. Thus, the common peptide sequence 33-37 in the positive peptide M1(28-37) and P1(33-42) is a key recognition sequence. Next, we studied the reactivity of some other synthetic peptides, in which some amino acids in the sequence 33-37 in peptide M1 changed to Ala. The peptides changing to Ala (PQ33-34AA) or (DGS35-37AAA) did not react with AIH-1 sera. Meanwhile, these AIH sera did not inhibit the glucuronidation of p-nitrophenol by UGT1A6, suggesting that the key sequence 33-37 might not be contained in active sites of glucuronidation by UGT1A6. In conclusion, sera from AIH-1 patients reacted with the amino acids in the sequence 33-37 (PQDGS) of the N-terminal of UGT1A6.

A new method to measure P-gp (ABCB1) activity.

We have developed an easy and sensitive method to measure P-glycoprotein (P-gp) activity using [gamma-(32)P]ATP and charcoal. This method utilizes the nature of adsorption of organic phosphate to charcoal. The standard assay method is as follows: the reaction mixture (20 microl) of 1 mM [gamma-(32)P]ATP (1 Ci/mol), 2.5 microg P-gp membranes, and the drug was incubated for 30 min, and 50 microl of 10% charcoal suspension in 0.1 M phosphate buffer at pH7.3 was then added to the mixture. The solution was centrifuged and the supernatant (20 microl) in duplicate containing inorganic (32)P was spotted onto filter paper, and radioactivity was measured by radio-image analyzer. This method can reduce the amount of P-gp membrane compared to the conventional method utilizing coloring of the inorganic phosphate-molybdate reaction. This method is also applicable to other ATP-binding cassette (ABC) transporters in phosphate buffer.

Induction of human UDP-glucuronosyltransferase 1A1 by cortisol-GR.

During the course of the study of UGT1A1 induction by bilirubin, we could not detect the induction of the reporter gene (-3174/+14) of human UGT1A1 in HepG2 by bilirubin (Mol. Biol. Rep. 31: 151-158 (2004)). In this report, we show the finding of the induction of the reporter gene of UGT1A1 by cortisol at 1 microM, a major natural cortico-steroid, with human glucocorticoid receptor (GR). RU486 of a typical GR antagonist at 10 microM inhibited the induction by cortisol from 5.9- to 1.8-fold. This result indicates that the induction by cortisol-GR is dependence on ligand-binding. This induction is caused by the UGT reporter gene itself, from the results of noinduction with control vector pGL2 (equal to pGV-C) in the presence of cortisol-GR. We confirmed that the induction of the reporter gene by cortisol is dependent on the position of proximal element (-97/-53) of UGT1A1. From this result, we concluded that the increase of corticosteroid in neonates must induce the elevation of UGT1A1 after birth and prevent jaundice. With the study of induction by corisol, we studied the influence of co-expression of PXR (pregnenolone xenobiotic receptor) with the UGT1A1 reporter gene and we could not find the induction of UGT1A1 expression in the presence of dexamethasone, rifampicin, or pregnenolone 16alpha-carbonitrile of the PXR ligands. These results suggest that the induction of UGT1A1 expression by GR is not mediated by PXR, unlike the induction of CYP3A4 through PXR.

Proximal HNF1 element is essential for the induction of human UDP-glucuronosyltransferase 1A1 by glucocorticoid receptor.

Previous study showed noinduction of the reporter gene (-3174/+14) of UGT1A1 in HepG2 by bilirubin, but induction by dexamethasone (DEX). This induction was enhanced seven-fold by the co-expression of human glucocorticoid receptor (GR) and was inhibited by a GR antagonist, RU486, indicating stimulation by DEX-GR. Meanwhile, we could not detect stimulation by beta-estradiol, phenobarbital or rifampicin (RIF) in the presence of GR. We investigated the position playing a role in this induction by GR in the promoter region of UGT1A1 using deletion mutants, and clarified the essential sequence (-75/-63) for the binding site of hepatocyte nuclear factor 1 (HNF1). However, GR did not bind directly to this sequence, because UGT-PE2 did not compete for binding to a glucocorticoid responsive element (GRE) probe in an electrophoretic mobility shift assay (EMSA) method. Labeled [(32)P]DNA probe of HNF1 binds with nuclear extracts as shown by the EMSA. This shift of the complex of probe-protein was not inhibited by unlabeled GRE but was inhibited by unlabeled HNF1 element. This shift was not influenced by the addition of anti-GR, but was super-shifted by the addition of anti-HNF1. GR did not stimulate the induction of HNF1, because we detected no-elevation of the mRNA level of HNF1 by reverse transcription-polymerase chain reaction (RT-PCR). Therefore, the induction of UGT1A1 by DEX-GR did not depend on the elevation of HNF1 but on the interaction of GR with HNF1 or the activation of HNF1 through the transcription of other proteins. Also given the lack of evidence of binding of DEX-GR to HNF1 in the EMSA, the data suggest that the mechanism of DEX-GRE effect on HNF1 is indirect by whatever mechanisms.

New horizon of MDR1 (P-glycoprotein) study.

MDR1 (once P-glycoprotein, now referred to as ABCB1) plays a role as a blood-brain barrier, preventing drug absorption into the brain, and is known to confer multiple drug resistance in cancer chemotherapy. MDR1 is composed of two repeated fragments, and there are six transmembrane domains (TMD) on the N-terminal of each repeat and a nucleotide (ATP) binding domain (NBD) on the C-terminal. These two repeats are dependent but cooperate as one functional molecule, with one pocket for excreting drugs. The 12 TM domains form a funnel facing the outside of cells, and NBD is in cytosol as a dimer. One NBD is composed of the Walker A, Q-loop, ABC-signature and the Walker B for phosphate binding of nucleotide. This tertiary structure of MDR1 is suggested from the structure of the NBD of histidine permease (HisP), clarified by x-ray crystallography. On the model of HisP, the NBD positions described above make a functional domain, and the same NBD structure is found on many other ABC transporters. An experiment with MDR1 gene knockout mice showed the high plasma AUC of drugs in mdr null mice [mdr1a(-/-)] and a high level in the brain, indicating that MDR1 has an efflux function (prevention of absorption) in the intestinal lumen and acts as a barrier of drug uptake in the brain, as well as has the function of urinary and biliary excretion of drugs. The transcription of MDR1 is dependent on two sites; the promoter site (-105/-100)(-245/-141) and the enhancer site (-7864/-7817). Autoantibody from autoimmune hepatitis patients weakly reacted with the extracellular peptide (aa314-aa328 between TM5 and 6) of MDR1 on the outside of the cell membrane, and did not react with peptides in the NBD and in the membrane-spanning region in TM5. There is an ambiguity about the function of MDR1 as GlcCer translocase.

Influence of synthetic and natural food dyes on activities of CYP2A6, UGT1A6, and UGT2B7.

Synthetic or natural food dyes are typical xenobiotics, as are drugs and pollutants. After ingestion, part of these dyes may be absorbed and metabolized by phase I and II drug-metabolizing enzymes and excreted by transporters of phase III enzymes. However, there is little information regarding the metabolism of these dyes. It was investigated whether these dyes are substrates for CYP2A6 and UDP-glucuronosyltransferase (UGT). The in vitro inhibition of drug-metabolizing enzymes by these dyes was also examined. The synthetic food dyes studied were amaranth (food red no. 2), erythrosine B (food red no. 3), allura red (food red no. 40), new coccine (food red no. 102), acid red (food red no. 106), tartrazine (food Yellow no. 4), sunset yellow FCF (food yellow no. 5), brilliant blue FCF (food blue no. 1), and indigo carmine (food blue no. 2). The natural additive dyes studied were extracts from purple sweet potato, purple corn, cochineal, monascus, grape skin, elderberry, red beet, gardenia, and curthamus. Data confirmed that these dyes were not substrates for CYP2A6, UGT1A6, and UGT2B7. Only indigo carmine inhibited CYP2A6 in a noncompetitive manner, while erythrosine B inhibited UGT1A6 (glucuronidation of p-nitrophenol) and UGT2B7 (glucuronidation of androsterone). In the natural additive dyes just listed, only monascus inhibited UGT1A6 and UGT2B7.

High levels of autoantibodies against drug-metabolizing enzymes in SLA/LP-positive AIH-1 sera.

Autoimmune hepatitis type 1 (AIH-1) is characterized by the detection of smooth muscle autoantibodies, antinuclear antibodies and antineutrophil cytoplasmic autoantibodies, and AIH-2 is characterized by the presence of autoantibodies against LKM, which contain drug-metabolizing enzymes. In this study, we measured the levels of drug-metabolizing enzymes in AIH-1 patients (ANA-positive). We exhaustively investigated the level of autoantibodies against major CYPs and UDP-glucuronosyltransferases of typical phase II drug-metabolizing enzymes, a transporter (MDR1), and NADPH-cytochrome P450 reductase in 4 patients with AIH-1 and 6 controls, as a case report. Two (Patients 3 and 4) of the AIH patients exhibited high levels of autoantibodies, while two (Patients 1 and 2) of the patients and the controls did not. The levels of autoantibodies against CYP2C19, CYP2D6, CYP2E1, UGT1A6 and human liver microsomes in Patients 3 and 4 sera were over 2(3) times the levels in Patient 1, Patient 2 and the control sera. Meanwhile, the levels of autoantibodies against CYP1A2, CYP2A6, CYP2C9, UGT2B7, MDR1 and NADPH-cytochrome P450 reductase were 2-2(2) higher in Patients 3 and 4 than in the other subjects. We found that the pattern of elevation in the Patient 3 serum was not parallel with that in Patient 4. Thus, we found high levels of autoantibodies against drug-metabolizing enzymes in AIH-1 patients.

Stimulation of transcriptional expression of human UDP-glucuronosyltransferase 1A1 by dexamethasone.

Human UDP-glucuronosyltransferase (UGT) 1A1 is only enzyme in the conjugation of bilirubin for prevention of hyperbilirubinemia and jaundice. Deletion or mutation of the UGT1A1 gene causes Crigler-Najjar syndrome or Gilbert's syndrome. We previously reported the functional promoter region for expression of UGT1A1 [Hepatology Research 9, 152-163 (1997)]. We investigated the influence of some drugs on the transient transfection assay of the luciferase reporter gene containing the 5'-promoter region -3174/+14 of UGT1A1 in HepG2 cells. Among drugs investigated, dexamethasone was the most effective at the range of concentration of 10-100 microM, whereas stimulation by beta-estradiol was not found. We also could not find stimulation by bilirubin of the endogenous main substrate for UGT1A1. Stimulation by dexamethasone was continued for 48 hr. The luciferase reporter gene containing the 5'-region of -97/+14 was induced by dexamethasone but the gene of the 5'-region -53/+14 was not. The region -97/-53 is essential for induction by dexamethasone. This region contains HNF1 element, therefore, we speculated that dexamethasone directly and/or indirectly stimulates UGT1A1 expression through this HNF1 region in the promoter region of UGT1A1. Thus, we clarified that UGT1A1 was induced by dexamethasone and the key position was the region (-97/-53) in UGT1A1 promoter.

Metabolic activation of 10-aza-substituted benzo[a]pyrene by cytochrome P450 1A2 in human liver microsomes.

We previously reported that 10-azabenzo[a]pyrene (10-azaBaP), a 10-aza-analog of BaP and an environmental carcinogen, showed greater mutagenicity than BaP in the Ames test using pooled human liver S9. To investigate the cytochrome P450 (CYP) isoform involved in the activation of 10-azaBaP to the genotoxic form, the mutagenicity of 10-azaBaP using nine individual donors' and pooled human liver microsome preparations was compared with each CYP activity. Induced revertants by 2.5 nmol per plate 10-azaBaP with 0.5 mg per plate human liver microsomal protein showed a large inter-individual variation (42-fold) among the nine donors. The number of induced revertants highly correlated with the CYP1A2-selective catalytic activity from each microsome preparation, and no correlation was observed with other CYP isoform-selective catalytic activities. Moreover, recombinant human CYP1A2 contributed to the mutagenicity of 10-azaBaP more markedly than recombinant human CYP1A1. These results suggest that CYP1A2 may be the principal enzyme responsible for the metabolic activation of 10-azaBaP in human liver microsomes. With regard to the proposal that BaP may be activated by human CYP1A1, our results suggest that the nitrogen-substitution at position-10 of BaP may cause the CYP enzyme-specificity in metabolic activation to change from CYP1A1 to CYP1A2.

Activation of the human Ah receptor by aza-polycyclic aromatic hydrocarbons and their halogenated derivatives.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor through which dioxins and carcinogenic polycyclic aromatic hydrocarbons cause altered gene expression and toxicity. Ten aza-polycyclic aromatic hydrocarbons (aza-PAHs), consisting of nitrogen substituted naphthalenes, phenanthrenes, chrysenes, and benzo[a]pyrenes (BaPs), were subjected to analysis of their structure-activity relationships as an AhR ligand by using a yeast AhR signaling assay, in which AhR ligand activity was evaluated as lacZ units. Most of the aza-PAHs showed similar or more potent AhR ligand activities than the corresponding parent PAHs. About a 100-fold increased in ligand activity was observed in 10-azaBaP compared with BaP. Halogen-substitution effects on AhR ligand activity in aza-polycyclic aromatics were also investigated with quinoline, benzo[f]quinoline (BfQ), benzo[h]quinoline (BhQ) and 1,7-phenanthroline (1,7-Phe). Position-specific induction of AhR ligand activity was observed in aza-tricyclic aromatic compounds, BfQ, BhQ, and 1,7-Phe, and the ratio of the ligand activities (lacZ units/microM) of monochlorinated and monobrominated aza-tricyclic aromatic compounds to those of the corresponding parent non-halogenated compounds ranged from 2.2- to 254-fold. Greatest enhancement of ligand activity was observed in 2-brominated BfQ (2-Br-BfQ), and its ligand activity was higher than that of BaP. These results suggest that even monohalogenation markedly enhances AhR ligand activity in aza-PAHs.

Study of inhibition of CYP2A6 by some drugs derived from quinoline.

CYP2A6 metabolizes coumarin to 7-hydroxycoumarin showing fluorescence, as measured by fluorometry. Firstly, we measured the inhibition of coumarin 7-hydroxylase of cDNA-expressed human CYP2A6 and in bovine liver microsomes, by quinoline and fluoroquinolines (FQ). Quinoline, 5-FQ, 6-FQ and 8-FQ inhibited activity but 3-FQ showed little inhibition. This suggests that the position 3 of quinoline is a recognition site for CYP2A6. We found similar patterns of coumarin 7-hydroxylase activity with human pooled liver microsomes. The level of CYP2A6 in human and bovine microsomes is the same as that detected by immunological titration with monoclonal antibody against CYP2A6. Secondly, we studied the inhibition of CYP2A6 with clinically used drugs of quinoline compounds, such as norfloxacin as an antibacterial agent, quinidine as an antiarrhythmic agent, quinine and chloroquine as antimalaria agents and rebamipide as an anti-ulcer agent. IC50 values (concentration producing 50% inhibition in activity) of norfloxacin, rebamipide and chloroquine at mM concentrations showed them to possess almost no inhibitory activity or influence on drug interaction. Meanwhile, the IC50 value of quinidine was 1.12 mM. The IC50 value of quinine was 160 microM with weak inhibition, suggesting that quinine, at a high dose, influences the metabolism of substrates for CYP2A6 by drug-drug interaction. These results also show that CYP2A6 discriminates the structure difference between the diastereoisomers quinidine and quinine.

pGp as the main product of bovine tRNA kinase.

One of the Ser-tRNAs, Ser-tRNA(Sec), is converted to Sec-tRNA(Sec) by Sec synthase. This Ser-tRNA(Sec) is also converted to phosphoser-tRNA(Sec) by tRNA kinase. In this study, we analyzed of the products of phosphorylation with tRNA kinase. [3H]Ser-tRNA(Sec) purified on Sephacryl S-200 was phosphorylated with [gamma-32P]ATP by tRNA kinase. The product [32P][3H]phosphoser-tRNA was purified on Sephacryl S-200 and hydrolyzed with ribonuclease T2. The chromatogram of this hydrolyzate on DEAE-cellulose in 7 M urea buffer showed four peaks. The first peak of the pass-through fraction was seryl-adenosine liberated from the 3'-terminal of the tRNA. The second peak, eluted before the third peak containing inorganic phosphate, was phosphoseryl-adenosine. The major compound in the fourth peak was pGp. As a control experiment, non-acylated tRNA(Sec) was used as a substrate of phosphorylation and the product was analyzed. The chromatogram of the digest with ribonuclease T2 showed no peak of phosphoseryl-adenosine, but a peak of pGp was seen with the peak of inorganic phosphate. Thus, the major product in the presence of tRNA kinase was pGp, and a small but significant proportion of the radioactivity was found as phosphoserine in the presence of seryl residue on the 3'-CCA terminal of tRNA(Sec). These results indicated that tRNA kinase phosphorylates not only Ser-tRNA to phosphoser-tRNA but also Gp of the 5'-termini of tRNA to pGp. This study gives a new role to mammalian tRNA kinase.

Effect of 10-aza-substitution on benzo[a]pyrene mutagenicity in vivo and in vitro.

Benzo[a]pyrene (BaP), an environmental carcinogen, shows genotoxicity after metabolic transformation into the bay-region diol epoxide, BaP-7,8-diol 9,10-epoxide. 10-Azabenzo[a]pyrene (10-azaBaP), in which a ring nitrogen is located in the bay-region, is also a carcinogen and shows mutagenicity in the Ames test in the presence of the rat liver microsomal enzymes. In order to evaluate the effect of aza-substitution on in vivo genotoxicity, BaP and 10-azaBaP were assayed for their in vivo mutagenicity using the lacZ-transgenic mouse (MutaMouse). BaP was potently mutagenic in all of the organs examined (liver, lung, kidney, spleen, forestomach, stomach, colon, and bone marrow), as described in our previous report, whereas, 10-azaBaP was slightly mutagenic only in the liver and colon. The in vitro mutagenicities of BaP and 10-azaBaP were evaluated by the Ames test using liver homogenates prepared from several sources, i.e. CYP1A-inducer-treated rats, CYP1A-inducer-treated and non-treated mice, and humans. BaP showed greater mutagenicities than 10-azaBaP in the presence of a liver homogenate prepared from CYP1A-inducer-treated rodents. However, 10-azaBaP showed mutagenicities similar to or more potent than BaP in the presence of a liver homogenate or S9 from non-treated mice and humans. These results indicate that 10-aza-substitution markedly modifies the nature of mutagenicity of benzo[a]pyrene in both in vivo and in vitro mutagenesis assays.