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Biochemical Pharmacology (v.72, #1)

Editorial Advisory Board (pp. iii).

Target-mediated drug disposition and dynamics by Donald E. Mager (pp. 1-10).

Nonlinear pharmacokinetics and pharmacodynamics may result from several capacity-limited processes and often represent complicating factors in characterizing the pharmacological properties of drugs. Target-mediated drug disposition (TMDD) corresponds to a special case wherein a significant proportion of a drug (relative to dose) is bound with high affinity to a pharmacological target, such that this interaction is reflected in the pharmacokinetic properties of the drug. Dose-dependent effects on apparent pharmacokinetic parameters may manifest, including the steady-state volume of distribution and total systemic clearance. Although a few small molecular weight compounds have been identified to exhibit TMDD, the incidence of TMDD is likely to increase particularly among emerging biotechnology pharmaceuticals. The goal of this commentary is to describe the basic tenets of TMDD and discuss several mathematical modeling approaches for characterizing this phenomenon. Whereas traditional pharmacokinetic/pharmacodynamic models assume that the amount of the drug–target complex is negligible relative to the total amount of drug in the body, integrated mechanism-based models of TMDD incorporate the binding and stoichiometry of drug–target binding. These models may be utilized to infer the time-course of inaccessible system variables, such as the in vivo density of the drug–target complex, and provide a suitable platform for ascertaining the apparent pharmacodynamic implications of TMDD.

Keywords: Abbreviations; ACE; angiotensin-converting enzyme; ACEI; ACE inhibitor; AUC; area under the plasma concentration–time curve; CL; total systemic drug clearance; EGF; epidermal growth factor; EPO; erythropoietin; G-CSF; granulocyte colony-stimulating factor; GP IIB/IIIa; glycoprotein αIIbβ3 platelet-surface receptor; IFN-β; interferon-beta; LIF; leukemia inhibitory factor; mAb; monoclonal antibody; PD; pharmacodynamics; PK; pharmacokinetics; TMDD; target-mediated drug disposition; t-PA; tissue plasminogen activator; TPO; thrombopoietin; VEGF; vascular endothelial growth factor; V; _ss; steady-state volume of distributionTarget-mediated drug disposition; Nonlinear pharmacokinetics; Pharmacodynamics; Mathematical modeling

Topoisomerase II beta levels are a determinant of melphalan-induced DNA crosslinks and sensitivity to cell death by M. Emmons; D. Boulware; D.M. Sullivan; L.A. Hazlehurst (pp. 11-18).

The role of topoisomerase (topo) II in DNA repair has yet to be fully elucidated. Current evidence suggesting a role for topo II in the repair of DNA damage has been obtained by using in vitro model systems or inferred from correlative data in drug resistant cell lines. In this study we directly examined the role of topo IIα and β in mediating the repair of melphalan-induced crosslinks in cellular DNA. To accomplish this, we used siRNA technology to knock down either topo IIα or β in human chronic myelogenous leukemia K562 and histiocytic lymphoma U937 cell line. Our data demonstrate that topo IIβ levels, (but not α), are a determinant of melphalan-induced crosslinks and sensitivity to melphalan. Furthermore, we show that knocking down topo IIβ inhibits the repair of melphalan-induced crosslinks in K562 cells. These studies represent the first direct evidence that topo IIβ participates in the repair of DNA damage induced by an alkylating agent in cellular DNA. Finally, these results suggest non-redundant roles for these two isoforms in mediating repair of DNA crosslinks.

Keywords: DNA repair; Topoisomerase II alpha; Topoisomerase II beta; Alkylating agents; Crosslinks; Drug resistance

Adenosine modulates vascular endothelial growth factor expression via hypoxia-inducible factor-1 in human glioblastoma cells by Stefania Merighi; Annalisa Benini; Prisco Mirandola; Stefania Gessi; Katia Varani; Edward Leung; Stephen Maclennan; Pier Andrea Borea (pp. 19-31).

Hypoxia appears to induce a program which shifts the cellular phenotype toward an increase in extracellular adenosine. Hypoxia-inducible factor-1 (HIF-1) is a key regulator of genes crucial to many aspects of cancer biology. Since in gliomas there is a strong correlation between HIF-1α expression, tumor grade and tumor vascularization, the aim of this study was to investigate whether adenosine may regulate HIF-1 in human glioblastoma cell lines. The results indicate that in the human hypoxic A172 and U87MG glioblastoma cell lines adenosine up-regulates HIF-1α protein expression via the A₃ receptor subtype. In particular, we investigated the effect of A₃ receptor antagonists on HIF-1 and vascular endothelial growth factor (VEGF) expression. We found that A₃ antagonists inhibit adenosine-induced HIF-1α and VEGF protein accumulation in the hypoxic cells. Investigations in the molecular mechanism showed that A₃ receptor stimulation activates p44/p42 and p38 MAPKs that are required for A₃-induced increase of HIF-1α and VEGF. Further studies are required to demonstrate the in vivo relevance of these observations with regard to the proposed role for adenosine as a key element in hypoxia and in tumors.

Keywords: Abbreviations; Act-D; actinomycin-D; Cl-IB-MECA; N; ⁶; (3-iodobenzyl)2-chloroadenosine-5′; N; -methyluronamide; GBM; glioblastoma multiforme; HIF-1; hypoxia-inducible factor-1; MAPK; mitogen-activated protein kinase; MEK; mitogen-activated protein kinase kinase; MRE 3008F20; 5; N; -(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)-pyrazolo-[4,3e]1,2,4-triazolo [1,5c] pyrimidine; MRE 3005F20; 5-[[(4-pyridyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine hydrochloride; RT-PCR; reverse transcription-PCR; siRNA; small interfering RNA; siRNA; _A3; small interfering RNA that targets A; ₃; receptor mRNA; siRNA; _HIF-1α; small interfering RNA that targets HIF-1α mRNA; VEGF; vascular endothelial growth factorAdenosine; A; ₃; receptors; Glioblastoma; HIF-1α; Hypoxia; VEGF

STAT3-independent inhibition of lysophosphatidic acid-mediated upregulation of connective tissue growth factor (CTGF) by cucurbitacin I by Angela Graness; Valeria Poli; Margarete Goppelt-Struebe (pp. 32-41).

Cucurbitacins are recognised as anti-tumour agents because of their interference with STAT3 signalling, but may also affect the integrity of the actin cytoskeleton. In the present study the effect of cucurbitacin I was investigated in fibroblasts. In these cells, cucurbitacin I interfered with lysophosphatidic acid (LPA) signalling. It inhibited tyrosine phosphorylation of focal adhesion proteins and induction of connective tissue growth factor (CTGF), a potent profibrotic protein. Inhibition of Src family kinases with PP2, but not the inactive analogue PP3, also interfered with LPA-mediated tyrosine phosphorylation and induction of CTGF. Jak2–STAT3 signalling seemed to be the connecting link, because CTGF induction was sensitive to AG490, an inhibitor of Jak2, and cucurbitacin I, an inhibitor of Jak2 and STAT3. However, LPA did not activate tyrosine phosphorylation of STAT3. Furthermore, cucurbitacin I was as effective in STAT3 knock out cells as in control cells. Therefore, the inhibitory effect of cucurbitacin I was not related to inhibition of STAT3.Immunocytochemical analysis of cucurbitacin I-treated cells revealed disassembly of F-actin fibres, reorganisation into F-actin patches and resolution of focal adhesions. The phenotypic changes resembled changes observed after treatment of the cells with cytochalasin D, which has been shown to interfere with CTGF induction. Concentrations of cucurbitacin I, which have been shown to target Jak2–STAT3 signalling, thus, profoundly affect the actin cytoskeleton and may therefore modulate cell morphology, migration, adherence and gene expression also in non-tumour cells.

Keywords: Abbreviations; CTGF; connective tissue growth factor; LPA; lysophosphatidic acid; STAT; signal transducers and activators of transcription; Jak; Janus kinaseActin cytoskeleton; Connective tissue growth factor; Lysophosphatidic acid; STAT3; Jak; Src kinase

Antagonism of peroxisome proliferator-activated receptor γ prevents high-fat diet-induced obesity in vivo by Ryosuke Nakano; Eiji Kurosaki; Shigeru Yoshida; Masanori Yokono; Akiyoshi Shimaya; Tatsuya Maruyama; Masayuki Shibasaki (pp. 42-52).

Peroxisome proliferator-activated receptor γ (PPARγ) has been reported to play an important role to regulate adiposity and insulin sensitivity. It is not clear whether antagonism of PPARγ using a synthetic ligand has significant effects on adipose tissue weight and glucose metabolism in vivo. The aim of this study is to examine the effects of a synthetic PPARγ antagonist (GW9662) on adiposity and glycemic control in high-fat (HF) diet-fed mice. First the properties of GW9662 as a PPARγ antagonist were estimated in vitro. GW9662 displaced [³H]rosiglitazone from PPARγ with K_i values of 13nM, indicating that the affinity of GW9662 for PPARγ was higher than that of rosiglitazone (110nM). GW9662 had no effect on PPARγ transactivation in cells expressing human PPARγ. Treatment of 3T3-L1 preadipocytes with GW9662 did not increase aP2 expression or [¹⁴C]acetic acid uptake. GW9662 did not recruit transcriptional cofactors to PPARγ. Limited trypsin digestion of the human PPARγ/GW9662 complex showed patterns of digestion distinct from those of rosiglitazone. This suggests that the binding characteristics between GW9662 and PPARγ are different from those of rosiglitazone. Treatment of HF diet-fed mice with GW9662 revealed that this compound prevented HF diet-induced obesity without affecting food intake. GW9662 suppressed any increase in the amount of visceral adipose tissue, but it did not change HF diet-induced glucose intolerance. These data indicate that antagonism of PPARγ using a synthetic ligand suppresses the increased adiposity observed in HF diet-induced obesity, and that a PPARγ antagonist could possibly be developed as an anti-obesity drug.

Keywords: Abbreviations; PPARγ; peroxisome proliferator-activated receptor γ; GW9662; 2-chloro-5-nitrobenzanilide; SRC-1; steroid receptor coactivator-1; SPA; scintillation proximity assay; LBD; ligand binding domain; GST; glutathione; S; -transferase; TZDs; 2,4-thiazolidinedionesPeroxisome proliferator-activated receptor γ; GW9662; High-fat diet; Adiposity; Receptor binding; Obesity

Paradoxical effects of the phage display-derived peptide antagonist IGF-F1-1 on insulin-like growth factor-1 receptor signaling by Stephanie A. Robinson; Steven A. Rosenzweig (pp. 53-61).

The insulin-like growth factor binding proteins (IGFBPs) represent a unique class of IGF antagonists regulating the bioavailability of the IGFs extracellularly. Accordingly, they represent an important class of proteins for cancer therapeutics and chemoprevention. IGF-F1-1 is a cyclic hexadecapeptide identified by high throughput phage display that binds to the IGFBP-binding domain on IGF-1. It acts as an IGFBP-mimetic, capable of inhibiting IGF-1 binding to the IGFBPs. To further examine the utility of IGF-F1-1 as an IGF-1 antagonist we tested its ability to inhibit IGFBP-2 and IGFBP-3 binding to IGF-1,¹²⁵I-IGF-1 binding to IGF-1Rs and to block IGF-1 induced Akt activation, cell cycle changes and [³H]thymidine incorporation in MCF-7 cells. These biological activities were inhibited by treatment with IGFBP-2, wortmannin or the IGF-1R tyrosine kinase inhibitor, NVP-AEW541, but not by IGF-F1-1. Our findings confirm previous studies indicating that IGF-F1-1 is a weak antagonist of IGF-1 binding to the IGFBPs and the IGF-1R and suggest that it does not effectively inhibit downstream events stimulated by IGF-1. We further demonstrated that IGF-F1-1 treatment of MCF-7 cells results in the paradoxical activation of Akt, S-phase transition and [³H]thymidine incorporation. These results suggest that IGF-F1-1 is a weak agonist, exhibiting mitogenic actions. IGF-F1-1 may act in conjunction with IGF-1 at the IGF-1R or independently of IGF-1 at the IGF-1R or another receptor.

Keywords: Abbreviations; Akt; protein kinase B (PKB) alpha isoform; BCA; bicinchoninic acid; BLOTTO; bovine lacto transfer technique optimizer; BSA; bovine serum albumin; Erk; extracellular-signal-regulated kinase; FBS; fetal bovine serum; Flt-1; fms-like tyrosine kinase-1; HRP; horse radish peroxidase; IGF-1; insulin-like growth factor 1; IGF-1R; IGF-1 receptor; IGFBP; IGF binding protein; IP; immunoprecipitation; IB; immunoblot; MAPK; MEK/mitogen-activated protein kinase; PBS; phosphate-buffered saline; PI 3-K; phosphatidylinositol 3-kinase; TBS; Tris-buffered saline; WCE; whole cell extractInsulin-like growth factor-1; Insulin-like growth factor binding proteins; Insulin-like growth factor antagonist; Structure–function; Breast cancer

Inhibition of homodimerization of Toll-like receptor 4 by curcumin by Hyung S. Youn; Shin I. Saitoh; Kensuke Miyake; Daniel H. Hwang (pp. 62-69).

Toll-like receptors play a key role in sensing microbial components and inducing innate immune responses. Ligand-induced dimerization of TLR4 is required for the activation of downstream signaling pathways. Thus, the receptor dimerization may be one of the first lines of regulation in activating TLR-mediated signaling pathways and induction of subsequent immune responses. LPS induces the activation of NF-κB and IRF3 through MyD88- or TRIF-dependent pathways. Curcumin, a polyphenol found in the plant Curcuma longa, has been shown to suppress the activation of NF-κB induced by various pro-inflammatory stimuli by inhibiting IKKβ kinase activity in MyD88-dependent pathway. Curcumin also inhibited LPS-induced IRF3 activation. These results imply that curcumin inhibits both MyD88- and TRIF-dependent pathways in LPS-induced TLR4 signaling. However, in TRIF-dependent pathway, curcumin did not inhibit IRF3 activation induced by overexpression of TRIF in 293T cells. These results suggest that TLR4 receptor complex is the molecular target of curcumin in addition to IKKβ. Here, we report biochemical evidence that phytochemicals (curcumin and sesquiterpene lactone) inhibit both ligand-induced and ligand-independent dimerization of TLR4. Furthermore, these results demonstrate that small molecules with non-microbial origin can directly inhibit TLRs-mediated signaling pathways at the receptor level. These results imply that the activation of TLRs and subsequent immune/inflammatory responses induced by endogenous molecules or chronic infection can be modulated by certain dietary phytochemicals we consume daily.

Keywords: Abbreviations; LPS; lipopolysaccharide; COX; cyclooxygenase; NF-κB; nuclear factor κB; MyD88; myeloid differential factor 88; TRIF; TIR domain-containing adapter inducing IFN-β; IFN; interferon; RIP1; receptor interacting protein 1; IKK; IκB kinase; TBK1; TANK-binding kinase 1; IRF3; IFN-regulatory factor 3; IRAK; IL-1 receptor-associate kinase; GFP; green fluorescent proteinToll-like receptor 4; LPS; Curcumin; Phytochemical; MyD88; TRIF

Dipeptidyl peptidase II and leukocyte cell death by Marie-Berthe Maes; Wim Martinet; Dorien M. Schrijvers; Pieter Van der Veken; Guido R.Y. De Meyer; Koen Augustyns; Anne-Marie Lambeir; Simon Scharpé; Ingrid De Meester (pp. 70-79).

Dipeptidyl peptidase (DPP) II (E.C. 3.4.14.2) is an intracellular protease that releases, preferably at acidic pH, N-terminal dipeptides from oligopeptides with Pro or Ala in the penultimate position. The natural substrates and the physiological role of DPPII remain unclear. The aim of the present study was to investigate the involvement of DPPII activity in different forms of cell death (apoptosis, necrosis and autophagy) in human leukocytes. We determined specific DPP activities in leukocytes. Compared to other subpopulations of peripheral blood mononuclear cells (PBMC), we observed relatively high DPPII specific activity in monocytic cells, opening new perspectives for further investigation of the DPPII functions. A second intriguing finding was that DPPII specific activity increased during necrosis, whereas induction of apoptosis or autophagy did not affect any of the dipeptidyl peptidase activities. Finally, we showed that inhibition of DPPII (>90%) using the in vitro applicable, highly potent ( K_i of 0.082±0.048nM) and selective DPPII inhibitor UAMC00039, did not induce any form of cell death in leukocytes. These data are of importance for a more precise interpretation of the in vitro and in vivo effects of other dipeptidyl peptidase inhibitors.

Keywords: Abbreviations; DPP; dipeptidyl peptidase; QPP; quiescent cell proline dipeptidase; CLL; chronic lymphocytic leukaemia; VbP; Val-boro-Pro; FAPα; fibroblast activation protein α; EBSS; Earle's balanced salt solution; CdA; 2-chloro-2′-deoxyadenosine; PI; propidium iodide; -pNA; -p-nitroanilideDPP; Quiescent cell proline dipeptidase; Val-boro-Pro; Apoptosis; Necrosis; Autophagy

Cytosolic heat shock proteins and heme oxygenase-1 are preferentially induced in response to specific and localized intramitochondrial damage by tetrafluoroethylcysteine by Han K. Ho; Yankai Jia; Kevin J. Coe; Qiuxia Gao; Catalin E. Doneanu; Zhonghua Hu; Theo K. Bammler; Richard P. Beyer; Nelson Fausto; Sam A. Bruschi; Sidney D. Nelson (pp. 80-90).

Previously, S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (TFEC) was shown to mediate cytotoxicity by covalently modifying a well-defined group of intramitochondrial proteins including aconitase, α-ketoglutarate dehydrogenase (αKGDH) subunits, heat shock protein 60 (HSP60) and mitochondrial HSP70 (mtHSP70). To investigate the cellular responses to this mitochondrial damage, microarray analysis of TFEC treated murine hepatocytes of the TAMH cell line was carried out. Results of these studies revealed a HSP response that was significantly stronger than other well-characterized hepatotoxicants including acetaminophen, diquat and rotenone. Specifically, cytosolic HSP25, HSP40, HSP70, HSP105 and microsomal HSP32 ( HO- 1) were strongly upregulated within the first few hours of TFEC treatment, while little change was observed among other HSPs that are predominantly localized in the mitochondria and endoplasmic reticulum (ER). Post-translational modification of HSP25 was also observed with the appearance of a unique DTT-resistant immunoreactive band at about 50kDa, a putative dimer. The biological significance of HSP responses to TFEC-induced toxicity were subsequently demonstrated using the “gain of function�? pretreatment: heat shock. Overall, we report an atypical HSP induction profile that does not conform to changes expected of a classical temperature shock. Furthermore, despite a well-defined intramitochondrial origin of toxicity, TFEC rapidly evokes an early and strong upregulation of cytosolic stress proteins. The cytoprotective effects of such HSP responses suggest a plausible role in modulating the progression of TFEC-induced cellular injury.

Keywords: Tetrafluoroethylcysteine; Heat shock proteins; Microarray; Cytotoxicity; Mitochondrial dysfunction; Heme oxygenase

Critical roles of AMP-activated protein kinase in the carcinogenic metal-induced expression of VEGF and HIF-1 proteins in DU145 prostate carcinoma by Minyoung Lee; Jin-Taek Hwang; Hee Yun; Eun Ju Kim; Min-Jung Kim; Sung-Soo Kim; Joohun Ha (pp. 91-103).

Epidemiological and experimental animal data indicate that exposure to both metals and metalloid species exacerbates the risk of human diseases, particularly cancers. Vascular endothelial growth factor (VEGF), which performs a primary function in both tumor progression and angiogenesis, is up-regulated due to exposure to an array of carcinogenic metals, but the mechanisms responsible for the metal activation remain somewhat poorly understood. Recently, we demonstrated that AMP-activated protein kinase (AMPK), which acts as an energy sensor, providing metabolic adaptation effects under ATP-deprived conditions, is critical for the expression of VEGF under oxygen- and glucose-deprived conditions. As carcinogenic metals are potent VEGF expression inducers, we hypothesized that AMPK would also play a crucial role in metal-induced VEGF expression. Here, we present evidence that carcinogenic metals such as arsenite, vanadate, and cobalt, induce AMPK activation and VEGF expression via several different mechanisms, and that AMPK is able to regulate the expression of VEGF mRNA in a hypoxia-inducible factor-1-dependent or -independent manner, depending on the metal applied. We also attempted to characterize the relevant signal transduction pathways in metal-induced VEGF expression and AMPK activation, as well as the role of reactive oxygen species within this context. Overall, our data suggest that AMPK is a critical regulatory component in metal-induced VEGF expression, which further implies its intrinsic involvement in metal-induced carcinogenesis.

Keywords: Abbreviations; ACC; acetyl-CoA carboxylase; AMPK; AMP-activated protein kinase; As; sodium arsenite; Co; cobalt chloride; DN; dominant negative; ELISA; enzyme-linked immunosorbent assay; ERK; extracellular-regulated kinase; GSH; reduced glutathione; HIF-1; hypoxia-inducible factor-1; JNK; c-jun-NH; ₂; -terminal kinase; MAPK; mitogen-activated protein kinase; NAC; N; -acetyl-cysteine; ROS; reactive oxygen species; RT; reverse transcriptase; Va; sodium orthovanadate; VEGF; vascular endothelial growth factor; WT; wild typeCarcinogenic metals; Vascular endothelial growth factor; Hypoxia-inducibale factor-1; AMP-activated protein kinase; Reactive oxygen species; Prostate carcinoma

Contractile mechanisms coupled to TRPA1 receptor activation in rat urinary bladder by Edinéia Lemos Andrade; Juliano Ferreira; Eunice André; João B. Calixto (pp. 104-114).

TRPA1 is a member of the transient receptor potential (TRP) channel family present in sensory neurons. Here we show that vanilloid receptor (TRPV1) stimulation with capsaicin and activation of TRPA1 with allyl isothiocyanate or cinnamaldehyde cause a graded contraction of the rat urinary bladder in vitro. Repeated applications of maximal concentrations of the agonists produce desensitization to their contractile effects. Moreover, contraction caused by TRPA1 agonists generates cross-desensitization with capsaicin. The TRP receptor antagonist ruthenium red (10–100μM) inhibits capsaicin (0.03μM), allyl isothiocyanate (100μM) and cinnamaldehyde (300μM)-induced contractions in the rat urinary bladder. The selective TRPV1 receptor antagonist SB 366791 (10μM) blocks capsaicin-induced contraction, but partially reduces allyl isothiocyanate- or cinnamaldehyde-mediated contraction. However, allyl isothiocyanate and cinnamaldehyde (10–1000μM) completely fail to interfere with the specific binding sites for the TRPV1 agonist [³H]-resiniferatoxin. Allyl isothiocyanate or cinnamaldehyde-mediated contractions of rat urinary bladder, which rely on external Ca²⁺ influx, are significantly inhibited by tachykinin receptor antagonists as well as by tetrodotoxin (1μM) or indomethacin (1μM). Allyl isothiocyanate-induced contraction is not changed by atropine (1μM) or suramin (300μM). The exposure of urinary bladders to allyl isothiocyanate (100μM) causes an increase in the prostaglandin E₂ and substance P levels. Taken together, these results indicate that TRPA1 agonists contract rat urinary bladder through sensory fibre stimulation, depending on extracellular Ca²⁺ influx and release of tachykinins and cyclooxygenase metabolites, probably prostaglandin E₂. Thus, TRPA1 appears to exert an important role in urinary bladder function.

Keywords: Allyl isothiocyanate; Cinnamaldehyde; Tachykinins; Prostaglandin E; ₂; Capsaicin; TRPV1; TRPA1

Expression of glutathione S-transferases in fetal lung and liver tissue from parental strains and F1 crosses between C57BL/6 and BALB/c F1 mice following in utero exposure to 3-methylcholanthrene by Mian Xu; Joseph E. Moore; Sandra Leone-Kabler; Thomas P. McCoy; Adam Swank; Garret B. Nelson; Jeffrey A. Ross; Alan J. Townsend; Mark Steven Miller (pp. 115-123).

GST isoforms have been extensively studied in adult tissues but little is known about the composition and levels of these enzymes in fetal tissues. As part of our ongoing studies to determine the potential role of metabolic enzymes in mediating the differential susceptibility of different strains of mice to lung tumorigenesis following in utero exposure to 3-methylcholanthrene (MC), we screened for GST enzyme activity and for expression of the individual GSTα, π, μ, and θ isoforms in murine fetal lung and liver tissues isolated from the parental strains and F1 crosses between C57BL/6 (B6) and BALB/c (C) mice. Using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, we found that treatment with MC had no effect on the levels of GST enzyme activity in either the fetal lung or liver in either of the two parental strains or their F1 crosses. Low levels of expression of each of the four enzymes were detected by Western blotting in both fetal lung and liver tissues in all four strains. A statistically significant 3.5-fold induction was observed only for GSTμ in the fetal lung of the parental strain of BALB/c mice 48h after exposure to MC. None of the other enzymes showed any significant differences in the levels of expression following exposure to MC. Although strain-specific differences in the expression of the GSTs that were independent of MC treatment were observed, they could not account for the differences previously observed in either the Ki- ras mutational spectrum or lung tumor incidence in the different strains of mice. Similar results were obtained when the maternal metabolism of MC was assayed in liver microsomal preparations. The results are consistent with previous studies showing low levels and poor inducibility of phase II enzymes during gestation, and demonstrate for the first time that all four of the major GST enzymes are expressed in fetal tissues. While the high inducibility of activating enzymes, such as Cyp1a1, and low, uninducible levels of phase II conjugating enzymes probably account for the high susceptibility of the fetus to transplacentally induced tumor formation, the results also suggest that factors other than metabolism may account for the strain-specific differences in susceptibility to carcinogen-mediated lung tumor induction following in utero exposure to chemical carcinogens.

Keywords: Abbreviations; AHH; aryl hydrocarbon hydroxylase activity; CDNB; 1-chloro-2,4-dinitrobenzene; Cyp; cytochrome P-450; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GST; glutathione; S; -transferase; MC; 3-methylcholanthrene; NAT; N; -acetyltransferases; PAH; polycyclic aromatic hydrocarbons; Pas; pulmonary adenoma susceptibility; UDPGT; uridine 5′-diphosphoglucuronosyltransferaseFetus; Glutathione; S; -transferases; 3-Methylcholanthrene; Lung tumors; Transplacental carcinogenesis

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Biochemical Pharmacology (v.72, #1)