The nucleotide excision repair protein XPC is essential for bulky DNA adducts to promote interleukin-6 expression via the activation of p38-SAPK.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, and many are potent carcinogens. Benzo[a]pyrene (B[a]P), one of the best-studied PAHs, is metabolized ultimately to the genotoxin anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE triggers stress responses linked to gene expression, cell death and survival. So far, the underlying mechanisms that initiate these signal transduction cascades are unknown. Here we show that BPDE-induced DNA damage is recognized by DNA damage sensor proteins to induce activation of the stress-activated protein kinase (SAPK) p38. Surprisingly, the classical DNA damage response, which involves the kinases ATM and ATR, is not involved in p38-SAPK activation by BPDE. Moreover, the induction of p38-SAPK phosphorylation also occurs in the absence of DNA strand breaks. Instead, increased phosphorylation of p38-SAPK requires the nucleotide excision repair (NER) and DNA damage sensor proteins XPC and mHR23B. Interestingly, other genotoxins such as cisplatin (CDDP), hydrogen peroxide and ultraviolet radiation also enhance XPC-dependent p38-SAPK phosphorylation. In contrast, anti-benzo[c]phenanthrene-3,4-dihydrodiol-1,2-epoxide, the DNA adducts of which are not properly recognized by NER, does not trigger p38-SAPK activation. As a downstream consequence, expression and secretion of the pro-inflammatory cytokine interleukin-6 is induced by BPDE and CDDP in vitro and by CDDP in the murine lung, and depends on XPC. In conclusion, we describe a novel pathway in which DNA damage recognition by NER proteins specifically leads to activation of p38-SAPK to promote inflammatory gene expression.

Phosphorylation of xenobiotic-metabolizing cytochromes P450.

The regulation of cytochromes P450 (CYPs) by induction mediated by xenobiotics is well known. Our team has discovered an additional important regulation of xenobiotic-metabolizing CYPs by phosphorylation. Individual CYPs are phosphorylated by different protein kinases, leading to CYP isoenzyme-selective changes in the metabolism of individual substrates and consequent profound changes in the control of mutagenic and cytotoxic metabolites. Some CYPs are phosphorylated by protein kinase C and some by the cyclic adenosine monophosphate (cAMP) dependent protein kinase A. We found that cAMP not only leads to drastic changes in the activity of individual CYPs, but also drastic changes in the nuclear localization of the CYP-related transcription factor Ah receptor (AHR). The consequences are very different from those of AHR nuclear translocation mediated by its classic ligands (such as dioxin and many polycyclic aromatic hydrocarbons) and may represent the long-sought physiological function of the AHR. The disturbance of this physiological function of AHR by extremely persistent high-affinity xenobiotic ligands such as dioxin may represent the most important contributing factor for their potent toxicity.

TCDD deregulates contact inhibition in rat liver oval cells via Ah receptor, JunD and cyclin A.

The aryl hydrocarbon receptor (AhR) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Activation of the AhR by TCDD leads to its dimerization with aryl hydrocarbon nuclear translocator (ARNT) and transcriptional activation of several phase I and II metabolizing enzymes. However, this classical signalling pathway so far failed to explain the pleiotropic hazardous effects of TCDD, such as developmental toxicity and tumour promotion. Thus, there is an urgent need to define genetic programmes orchestrated by AhR to unravel its role in physiology and toxicology. Here we show that TCDD treatment of rat liver oval cells leads to induction of the transcription factor JunD, resulting in transcriptional upregulation of the proto-oncogene cyclin A which finally triggers a release from contact inhibition. Ectopic expression of cyclin A in confluent cultures overcomes G(1) arrest, indicating that increased cyclin A levels are indeed sufficient to bypass contact inhibition. Functional interference with AhR-, but not with ARNT, abolished TCDD-induced increase in JunD and cyclin A and prevented loss of contact inhibition. In summary, we have discovered a novel AhR-dependent and probably ARNT-independent signalling pathway involving JunD and cyclin A, which mediates TCDD-induced deregulation of cell cycle control.

Phosphorylation of cytochromes P450: first discovery of a posttranslational modification of a drug-metabolizing enzyme.

Cytochromes P450 (CYP) are important components of xenobiotic-metabolizing monooxygenases (CYP-dependent monooxygenases). Their regulation by induction, most commonly by transcriptional activation, mediated by xenobiotics, normally substrates of the corresponding CYP, is well known and has been widely studied. Our team has discovered an additional important regulation of xenobiotic-metabolizing CYPs pertaining to posttranslational modification by phosphorylation. Individual CYPs are phosphorylated by different protein kinases, leading to CYP isoenzyme-selective changes in the metabolism of individual substrates and consequent drastic changes in the control of genotoxic metabolites. Best studied are the CYP phosphorylations by the cAMP-dependent protein kinase A. Most recently, we discovered that cAMP not only leads to drastic changes in the activity of individual CYPs, but also to drastic changes in the nuclear localization of the CYP-related transcription factor Ah receptor (AHR). The consequences are very different from those of AHR nuclear translocation mediated by the classical ligands (enzyme inducers such as dioxin) and are likely to represent the long-sought physiological function of the AHR, its persistent disturbance by long-lived ligands such as dioxin may well be the reason for its high toxicity.

cAMP-dependent phosphorylation of CYP2B1 as a functional switch for cyclophosphamide activation and its hormonal control in vitro and in vivo.

An important feature of cytochrome P450 (CYP) 2B1 is its high ability to convert the prodrug cyclophosphamide (CPA) to therapeutically cytotoxic metabolites, resulting in interstrand DNA-cross-linking and cell death. We have examined whether and how the phosphorylation of CYP2B1 influences CPA metabolic activation in vitro and in vivo. We found first that only part of the total CYP2B1 pool undergoes phosphorylation. This part is fully inactivated. Second, phosphorylation of CYP2B1 in intact hepatocytes reduced by up to 75% toxification of CPA to mutagenic metabolites (totally dependent on the same preferentially CYP2B-catalyzed 4-hydroxylation of CPA as is the generation of highly cytotoxic species). Third, the phosphoacceptor-serine 128 of CYP2B1 in the consensus sequence for interaction with the protein kinase A represents an on/off switch for the activation of CPA depending on the phosphorylation conditions in the cell. Fourth, evidence is presented that the above-described events also occur in vivo. In conclusion, a successful therapy with CPA, helped by forced expression of CYP2B1 in tumor cells (as recently proposed) will, in addition, be profoundly modified by its phosphorylation status.

Induction of DNA single-strand breaks by 131I and 99mTc in human mononuclear blood cells in vitro and extrapolation to the in vivo situation.

The radionuclides (131)I and (99m)Tc are frequently used for therapy of benign and malignant thyroid disease ((131)I) and for diagnosis of thyroid and other diseases ((99m)Tc). However, the levels of DNA single-strand breaks (SSBs) induced in cells of patients after administration of (131)I and (99m)Tc are not known. In this study, we measured the number of SSBs per cell induced by (131)I and (99m)Tc in vitro, extrapolated the results to the clinical situation, and assessed their biological relevance by comparing levels of SSBs induced after therapeutic administration of (131)I and (99m)Tc to those induced by endogenous processes or by occupational exposure to genotoxic substances. A linear dose-response relationship between the radioactivity concentrations of (131)I and (99m)Tc and SSBs in human mononuclear blood cells (determined by alkaline elution) was obtained after incubation at 4 and 37 degrees C. At 4 degrees C, where almost no repair of SSBs takes place, (131)I and (99m)Tc induced 81 and 7 SSBs per cell per hour/(MBq/ml), respectively. At 37 degrees C, only 20 and 1.6 SSBs per cell per hour/(MBq/ml) were observed after incubation with (131)I and (99m)Tc. To estimate the induction of SSBs in vivo in cells of patients after administration of 3700 MBq (131)I (oral) or 60 MBq (99m)Tc (i.v.), the rates of induction of SSBs obtained in vitro were extrapolated to the concentrations of (131)I and (99m)Tc measured in blood of patients. The total number of SSBs (mean +/- standard deviation) accumulated after oral administration of 3700 MBq (131)I up to 70 h after administration was calculated as 200 +/- 59 SSBs/cell. After administration of 60 MBq (99m)Tc (i.v.), 0.032 +/- 0.009 SSBs per cell (total SSBs up to 2 h after administration) were cumulated. The induction of SSBs by endogenous processes (estimated 2,000 SSBs per cell per hour) and by occupational exposure to genotoxic substances (125-430 SSBs per cell) has been estimated in earlier studies. In conclusion, the frequency of SSBs induced by thyroid diagnosis with 60 MBq (99m)Tc is approximately 5 orders of magnitude smaller than the frequency of spontaneous SSBs and thus is most probably without biological relevance. Since the frequency of induction of SSBs by therapy with (131)I (3700 MBq) is about 6000-fold higher compared to thyroid diagnosis by (99m)Tc, its biological relevance is more difficult to assess. Nevertheless, the number of SSBs induced by therapy with (131)I is substantially lower than that induced by endogenous processes.

Glutathione S-transferase T1 and M1 gene defects in ovarian carcinoma.

Glutathione S-transferases (GSTs) M1 and T1 are known to be polymorphic in humans. Both polymorphisms are due to gene deletions, which are responsible for the existence of null genotypes. The gene defect of GSTT1 has been reported to be associated with an increased risk of myelodysplastic syndromes, astrocytoma and meningioma. A lack of GSTM1 was associated with tobacco smoke-induced lung and bladder cancer. In this study we examined whether the GSTT1 and/or GSTM1 homozygous null genotypes were associated with an increased risk of ovarian cancer using a multiplex polymerase chain reaction protocol. The GSTT1 null genotype was observed in 14% of the control subjects that had never suffered from neoplastic disease (n = 115) and in 16% of the patients affected with ovarian cancer (n = 103, OR 0.87, 95% CI 0.39-1.92, P = 0.73). A lack of GSTM1 was observed in 38% of the control subjects and in 46% of the patients (OR 0.77, 95% CI 0.44-1.32). This difference was not significant (P = 0.34). Similarly, no significant differences were obtained if GSTT1 and/or GSTM1 null genotypes were analyzed in subgroups of control subjects and ovarian cancer patients between the ages of 20-40, 41-70 and 71-90 years and in individuals with a positive family history of neoplastic disease. GSTT1 and/or GSTM1 null genotypes were not significantly associated with the histologic type and grade or FIGO (International Federation of Gynecology and Obstetrics) stages of the ovarian carcinomas. In conclusion, GSTT1 and/or GSTM1 null genotypes are not markers for an increased risk of ovarian cancer.

Differential modulation of CYP2E1 activity by cAMP-dependent protein kinase upon Ser129 replacement.

Many toxic compounds are activated by cytochrome P450 (CYP) 2E1 to reactive metabolites, which represents a potential hazard for cellular homeostasis. Therefore knowledge about CYP2E1 regulation could be of great biological importance. It has been shown that CYP2E1 is controlled transcriptionally and post-translationally by phosphorylation. In the present study we investigated the role of serine-129 (Ser129) in the protein kinase A (PKA) recognition sequence motif Arg-Arg-Phe-Ser129. To gain further insights into the possible relevance of Ser129 for CYP2E1 function, Ser129 was replaced by alanine (Ala) or glycine (Gly) by site-directed mutations of the cDNA coding for CYP2E1. The mutant cDNAs were transfected into Chinese hamster lung fibroblast V79 cells. Despite the mutation in the PKA phosphorylation motif, all strains produced catalytically active CYP2E1. However, there was a marked change in the substrate preference: The Gly129-containing strains hydroxylated p-nitrophenol (PNP) to a markedly higher extent than the wild-type cDNA-containing cells, while they demethylated N-nitrosodimethylamine (NDMA) to a markedly lower extent than the wild-type cells. All the strains activated NDMA to mutagenic products. Treatment with the membrane-permeating cAMP derivative db-cAMP reduced markedly both the PNP hydroxylase and the NDMA demethylase activities as well as the mutation frequency induced by NDMA in the Ser129-containing strain. This decrease in activity was not accompanied by a decrease in CYP2E1 content. In addition, the catalytic activities of CYP2E1 were decreased in microsomes from rat hepatocytes treated with db-cAMP. Also in this case, the decrease in activities was not accompanied by a decrease in enzyme protein. These findings argue that involvement of Ser129 and its phosphorylation is not in determining CYP2E1 protein level, but rather in controlling its catalytic activity. In contrast, in the strains containing Ala129 or Gly129, treatment with db-cAMP caused a marked increase in both PNP hydroxylase and NDMA demethylase. In these strains a similar db-cAMP-mediated increase was also observed in the mutation frequency, resulting from the treatment with the promutagen NDMA, which is activated by CYP2E1. Our results show that CYP2E1 in V79 cells responds in two separate ways to db-cAMP exposure depending on the amino acid residue present in the PKA recognition sequence. The enzyme is committed to a negative regulation by db-cAMP if Ser129 is the target amino acid for PKA, leading to a decrease in the metabolic activation to mutagenic and carcinogenic species. On the other hand, Ala129 or Gly129 substitution directed CYP2E1 toward a positive regulation by increasing its catalytic activities and metabolic activation to mutagenic intermediates in the presence of db-cAMP. We also obtained evidence that cAMP-mediated downregulation of wild-type (Ser129) CYP2E1 was not accompanied by its destruction but instead by its stabilization, which shows that Ser129 is not involved in CYP2E1 degradation but dictates requirements for its specific activities.

Increase in DNA single-strand break rejoining by continuous exposure of human mononuclear blood cells to radioiodine ((131)I) in vitro.

Radioiodine ((131)I) induced a dose- and time-dependent increase in DNA single-strand breaks (DNA-ssb) in human (G0) mononuclear blood cells (MNC) in vitro. Incubation of MNC with 22MBq (131)I/ml at 4 degrees C caused a linear, time-dependent induction of DNA-ssb (increase in elution rate: 24.7 x 10(-3) h(-1) per 100 min incubation with (131)I). However, if MNC were incubated at 37 degrees C a decrease in the slope of the time effect curve was observed after about 300 min incubation with 22 or 30 MBq (131)I/ml. The goodness of fit of different regression models was assessed by Akaike's Information Criterion (AIC). The best fit was obtained for a non-linear model (y=a+bx+cx(0.5); AIC=53.5; where x is incubation time and y is elution rate), whereas other models including the linear regression model y=a+bx; AIC=38.6) were worse. As the total induction of DNA-ssb at 4 degrees C was constant with time, the decrease in the slope of the time effect curve (DNA-ssb versus time) at 37 degrees C can be interpreted as an increase in rejoining of DNA-ssb. Inhibition of both RNA and protein synthesis clearly increased the extent of DNA-ssb observable after incubation with (131)I. Thus, during continuous exposure of MNC to (131)I, proteins were synthesized which rejoined DNA-ssb. However, incubation of MNC with (131)I (44 MBq/ml) at 37 degrees C under conditions expected to lead to inhibition of RNA and/or protein synthesis still resulted in a decrease of the slope of the time effect curve, indicating a stimulation of DNA-ssb rejoining. Thus, we favour the hypothesis that the increase in the activity of DNA-ssb rejoining, besides de novo synthesis of repair enzymes, is also caused by a post-translational stimulation of DNA-repair enzymes and that this stimulation possibly is mediated by DNA-fragments.

Analysis of DNA single-strand breaks in human venous blood: a technique which does not require isolation of white blood cells.

For DNA strand break analysis in human white blood cells, usually metrizoate-Ficoll centrifugation is used to isolate mononuclear cells. This procedure is time-consuming and requires at least 20 ml of blood per sample. Therefore, we developed a technique which does not require isolation of white blood cells prior to DNA strand break analysis by alkaline elution (direct method). The sensitivity of this new technique was compared to that of the standard method, which includes isolation of mononuclear blood cells. A statistically significant increase in sensitivity was observed using the direct method. After in vitro gamma-irradiation of venous blood, an increase in the elution rate of 7.7 x 10(-3) hr(-1)/Gy was detected if mononuclear blood cells were isolated compared to 10.5 x 10(-3) hr(-1)/Gy with the new technique (P < 0.05). Incubation of venous blood with ethylene oxide for 1 hr caused an increase in the elution rate of 5.8 x 10(-3) hr(-1)/mM ethylene oxide for the standard and 12 x 10(-3) h(-1)/mM for the direct method (P < 0.05). DNA single-strand breaks were detected in blood cells of 10 persons without any apparent genotoxic exposure. A mean normalized elution rate of 1.30 +/- 0.38 (95% confidence interval) was detected in isolated mononuclear blood cells, and a similar mean normalized elution rate of 1.41 +/- 0.50 was obtained using the direct method. The difference was not statistically significant. Five patients treated with a combination chemotherapy consisting of cyclophosphamide (750 mg/m2 i.v.), doxorubicin (50 mg/m2 i.v.), vincristine (1.4 mg/m2 i.v.), and prednisolone (100 mg/m2 p.o.) for non-Hodgkin's disease were analyzed for DNA single-strand breaks before and 16-18 hr after the application of chemotherapy. Increases in mean elution rate of 68% and 116% were detected using the standard and the direct methods, respectively. For the direct method, only 3 ml of venous blood were sufficient for analysis of one sample, compared to 25 ml needed if mononuclear cells were isolated, and about 4 hr of work per assay can be saved.

Control of the mutagenicity of aromatic amines by protein kinases and phosphatases. I. The protein phosphatase inhibitors okadaic acid and ortho-vanadate drastically reduce the mutagenicity of aromatic amines.

The role of protein kinase C and protein phosphatases was examined in the control of mutagenic metabolites of aromatic amines. Various metabolic activating systems derived from rat liver were treated with: 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C modulator; okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatases (PP1 and PP2A); and ortho-vanadate (OV), an inhibitor of tyrosine phosphatases. TPA used over a wide concentration range (10(-9)-10(-6) M) did not affect the bacterial mutagenicity of the aromatic amines and of the aromatic amide investigated, 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene (2AAF). At the molecular level, TPA did not affect the function of cytochrome P450s 1A1 or 1A2, which are known key factors for the activation and inactivation of aromatic amines/amides. By contrast the OA and OV treatment of rat hepatocytes, rat liver homogenate, fraction S9 and the nuclear fraction drastically reduced (by > 80%) the mutagenicity of the aromatic amines/amide investigated. This is by far the most pronounced change in genotoxicity observed to date via modulation of phosphorylation. Whilst the mutagenicity of the primary toxication product 2-N-OH-acetylaminofluorene (2-N-OH-AAF) in the presence of exogenous activating systems (hepatocytes, S9-fraction, nuclear fraction) was also reduced by OV, OA had no influence. Thus the tyrosine protein phosphatase inhibitor and the serine/threonine protein phosphatase inhibitor influence the genotoxicity of aromatic amines/amides on different levels. Moreover, this shows that the drastic reduction in mutagenicity by OA was due to its influence on a step prior to the presence of the primary toxication product 2-N-OH-AAF. This reduction could be due to changes in the activity of cytochrome P4501A1 and/or 1A2. However, no incorporation of 32P-labelled phosphate from intracellularly prelabelled [32P]-ATP into cytochromes P450 1A1 or 1A2 nor any change in their catalytic activities was observed in the presence of OA. Furthermore, a phosphorylation dependent change in the function of P-glycoprotein (known for its role in the transport of diverse xenobiotic substances and their metabolites) was shown not to contribute to the observed decrease in mutagenicity. Our results reveal an important role for protein phosphatase 1 and/or 2A and tyrosine phosphatase(s) in the control of the genotoxicity of aromatic amines and amides. However, the present study does not distinguish between effects mediated by individual proteins affected by these protein phosphatases.

Control of the mutagenicity of arylamines by protein kinases and phosphatases: II. Lack of response of rat liver N-acetyl transferases to phosphorylation modulators.

Treatment of rat hepatocytes with the phosphatase inhibitors okadaic acid or ortho-vanadate had led to an 80% decrease in the bacterial mutagenicity of several aromatic amines metabolically activated by these hepatocytes. This is the most dramatic change yet demonstrated in mutagenicity by phosphorylation modulation. However, incorporation of phosphate into and catalytic activity of cytochromes P450 (CYP) 1A1 and 1A2, the major catalysts for the first step in the toxication of aromatic amines, were unchanged. We therefore investigated whether changes in the phosphorylation status would influence the activities of the N-acetyltransferases NAT1 and/or NAT2, being responsible for one of the two major pathways leading to the ultimate mutagens, the reactive esters which are derived from the N-hydroxylated metabolites of aromatic amines. Hepatocytes were derived from the livers of rats pretreated with CYP1A1/1A2 inducers and from untreated rats using conditions under which the phosphorylation-dependent drastic decrease of the arylamine mutagenicity was observed. Treatments were exposure to 1 mM dibutyryl-cAMP (protein kinase A stimulator), 100 nM okadaic acid or 20 nM calyculin A (preferential inhibitors of serine/threonine phosphatases PP2A and PP1, respectively), 2 mM ortho-vanadate (inhibitor of tyrosine phosphatases), and 50 mM NaF (stimulator of adenylate cyclase and non-specific inhibitor of protein phosphatases). None of the phosphorylation modulators led to a significant change in NAT1 or NAT2 activities. This was true for hepatocytes from rats which had been pretreated with inducers for CYP1A1 and CYP1A2 as well as from untreated rats. The inducers led to the expected increases in CYP1A1 and CYP1A2 but the NAT1 and NAT2 activities remained unchanged. Our study shows that the N-acetyl transferases NAT1 or NAT2, the catalysts responsible for the formation of the highly reactive N-acetoxy derivatives of N-hydroxylated aromatic amines, are not responsible for the drastic decrease in arylamine genotoxicity after treatment of the metabolizing system with protein phosphatase inhibitors. The data also show that NAT1 and NAT2 are not regulated by the classical xenobiotic metabolizing enzyme inducers nor by any of the phosphorylation modulators used.

Mechanism-based predictions of interactions.

Exposure to more than one toxic compound is common in real life. The resulting toxic effects are often more than the simple sum of the effects of the individual compounds. It is unlikely that it will ever be possible to test all combinations. It is therefore highly desirable to improve or develop means for reasonably approximating predictions of interactions. In order to be valid and extrapolatable, these predictions are most promising if they are mechanism-based. Examples will be given for possibilities of mechanism-based predictions of interactions which exceed trivialities of simple increases by enzyme induction of enzymatic rates of a given biotransformation pathway leading to a toxic metabolite. Instead, examples will be provided where competition between various enzymes for shunting the same substrate into divergent pathways can lead to predictable dramatic changes in toxicity by shifting the metabolic routes under conditions of no significant changes of overall metabolism. Further examples are given on predictable interactions between chemicals which need bioactivation for exerting their toxicity and chemicals which effect hormonal status and other endogenous factors which in turn modify enzymes involved in the control of toxic metabolites.

Molecular and cellular basis for adequate metabolic design of genotoxicity studies.

Genotoxic species and metabolites are usually under the control of a complex set of activating, inactivating and precursor sequestering enzymes. These enzymes differ greatly between test systems, animal species and man. An adequate metabolic design of genotoxicity studies requires careful attention to factors such as: Dilution of cofactors in in vitro tests which are present in much higher concentrations in the intact cell; Induction in high dose carcinogenicity bioassays of enzymes, which are constitutively not expressed and not induced at such doses of the compound, which occur in the situations of the practical use of the compound; Modifications of control enzymes, which are effected by hormones or other endogenous factors, which are differently influenced by high dose (bioassay) versus moderate dose (real exposure) or by in vivo (endocrine regulation) versus in vitro (no endocrine regulation) conditions.

Modulation of the control of mutagenic metabolites derived from cyclophosphamide and ifosfamide by stimulation of protein kinase A.

The phosphorylation of the 2 major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in intact hepatocytes by the action of the membrane-permeating cAMP derivative N6,O2'-dibutyryl-cAMP. Under these conditions cyclophosphamide and ifosfamide (which are known to be activated by cytochrome P450 IIB1) were investigated for mutagenicity in Salmonella typhimurium TA1535 and TA100 and for cytotoxicity in TA1535. Cyclophosphamide and ifosfamide were transformed to mutagenic and cytotoxic metabolites by the hepatocytes. The activation of both drugs to mutagens was markedly reduced after pretreatment of the hepatocytes with the membrane-permeating cAMP derivative N6,O2'-dibutyryl-cAMP. Cyclophosphamide and ifosfamide activation were reduced to 51% and 38% of unstimulated controls respectively, when hepatocytes were incubated for 1 h with N6,O2'-dibutyryl-cAMP in the presence of the phosphodiesterase inhibitor theophylline, and Salmonella typhimurium TA1535 was used. A marked reduction in mutagenicity of cyclophosphamide (35% compared with unstimulated controls) was also observed under different experimental conditions, namely after pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP for 1.5 h without theophylline and using Salmonella typhimurium TA100 as target strain. Continued presence of the cytochrome P450 IIB1 and P450 IIB2 inducer phenobarbital in the stimulation medium increased the mutagenicity of cyclophosphamide and led to an even more marked reduction of mutagenicity by pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP and theophylline. In order to investigate whether the observed changes were metabolism-related, the ifosfamide metabolite ifosfamide mustard which does not require metabolic activation by cytochrome P450 was studied under the same conditions. Its mutagenicity was indistinguishable after incubation with N6,O2'-dibutyryl-cAMP-treated or with unstimulated hepatocytes. Also the metabolic formation of cytotoxic metabolites from cyclophosphamide and ifosfamide but not that of ifosfamide mustard was markedly decreased by pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP and theophylline. Thus the stimulation of protein kinase A in intact cells has important consequences for the control of genotoxic and cytotoxic metabolites and represents a fast and short-term regulation of it.

Toxicological implications of enzymatic control of reactive metabolites.

Many foreign compounds are transformed into reactive metabolites, which may produce genotoxic effects by chemically altering critical biomolecules. Reactive metabolites are under the control of activating, inactivating and precursor sequestering enzymes. Such enzymes are under the long-term control of induction and repression, as well as the short-term control of post-translational modification and low molecular weight activators or inhibitors. In addition, the efficiency of these enzyme systems in preventing reactive metabolite-mediated toxicity is directed by their subcellular compartmentalization and isoenzymic multiplicity. Extrapolation from toxicological test systems to the human requires information of these variables in the system in question and in man. Differences in susceptibility to toxic challenges between species and individuals are often causally linked to differences in these control factors.

Phosphorylation of cytochrome P450 isoenzymes in intact hepatocytes and its importance for their function in metabolic processes.

Recent data show that besides the well-known long-term regulation of cytochrome P450-dependent monooxygenase activity by induction there also exists a fast regulation by phosphorylation. This phosphorylation occurs when purified cytochromes P450 are combined with purified protein kinases, and also in intact cells. This process is donor- and acceptor-selective leading to phosphorylation of defined isoenzymes by defined protein kinases. This in turn leads to fast and marked changes in metabolism which are selective for given substrates and regio- and stereo-selective for given positions. This in turn is selectively and differentially influenced by the individual control of the protein kinase in question.