Early Childcare and Education in a Post-Industrial Landscape: Inequalities in Proximity to Active and Relic Manufacturing in Metropolitan Providence, Rhode Island.

Children are uniquely vulnerable to environmental health risks associated with industrial contamination, and early childcare and education (ECE) facilities are important sites for potential exposure to environmental contaminants. Emerging research on historic urban industry has additionally demonstrated that urban environmental risk accumulates historically and spatially across urban landscapes. Accordingly, this study pairs cross-sectional data on licensed childcare facilities with longitudinal manufacturing site data in Providence, Rhode Island. We use these data to investigate the proximity of ECE facilities to active and relic manufacturing sites, controlling for a range of organizational- and tract-level characteristics. Results show that type of childcare facility (center-based vs. in-home) and language of instruction (Spanish vs. English) are important predictors of children's proximity to industrial lands, past and present. These findings indicate that Spanish-speaking children in Providence may experience a "double jeopardy" in the form of disproportionate legacy environmental hazards at ECE as well as at home-suggesting that the historical nature of urban industrial land use is an important mechanism of environmental inequality for young children.

Regulation of progesterone signaling during pregnancy: implications for the use of progestins for the prevention of preterm birth.

Preterm birth is a major cause of neonatal morbidity and mortality. Progesterone plays a critical role in suppressing the inflammatory signals that would induce parturition prior to term. Progesterone signaling is regulated in a variety of ways during pregnancy. Endocrine production of high levels of progesterone by the placenta ensures the availability of high levels of progesterone throughout pregnancy. Paracrine regulation of progesterone metabolism in target tissues, particularly the myometrium and cervix, also determines the amount of progesterone ligand available. Progesterone metabolism can also lead to the formation of metabolites that contribute to its effects. In particular, 5ß-dihydroprogesterone formation by aldo-keto reductase 1D1 appears to play an important role in maintaining uterine quiescence. Progesterone signaling can also be regulated at the receptor level through changes in the relative expression of the nuclear progesterone receptor isoforms, reduced expression of membrane receptors, and changes in the expression levels of coactivators and/or corepressors, including nuclear factor κB. Progesterone and 17α-hydroxyprogesterone caproate (17OH-PC) have recently been shown to reduce preterm births in women with previous preterm birth or shortened cervix. It is important to realize that these two progestins are likely to act in significantly different ways, which will likely influence their efficacy. The structural differences and resistance to metabolism exhibited by 17OH-PC means that it will be unable to activate some of the pathways that progesterone activates, but that it also will not be subject to paracrine inactivation. The fact that progesterone therapy works for maintaining pregnancy in some women, indicates that for those women insufficient levels of progesterone ligand in target tissues is a determining factor in early parturition, despite high levels of circulating progesterone. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

Development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization/selected reaction monitoring/mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of keto-androgens in human serum.

Prostate cancer is the most frequently diagnosed form of cancer in males in the United States. The disease is androgen driven and the use of orchiectomy or chemical castration, known as androgen deprivation therapy (ADT) has been employed for the treatment of advanced prostate cancer for over 70 years. Agents such as GnRH agonists and non-steroidal androgen receptor antagonists are routinely used in the clinic, but eventually relapse occurs due to the emergence of castration-resistant prostate cancer. With the appreciation that androgen signaling still persists in these patients and the development of new therapies such as abiraterone and enzalutamide that further suppresses androgen synthesis or signaling, there is a renewed need for sensitive and specific methods to quantify androgen precursor and metabolite levels to assess drug efficacy. We describe the development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization selected reaction monitoring mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of serum keto-androgens and their sulfate and glucuronide conjugates using Girard-T oxime derivatives. The method is robust down to 0.2-4pg on column, depending on the androgen metabolite quantified, and can also quantify dehydroepiandrosterone sulfate (DHEA-S) in only 1µL of serum. The clinical utility of this method was demonstrated by analyzing serum androgens from patients enrolled in a clinical trial assessing combinations of pharmacological agents to maximally suppress gonadal and adrenal androgens (Targeted Androgen Pathway Suppression, TAPS clinical trial). The method was validated by correlating the results obtained with a hydroxylamine derivatization procedure coupled with tandem mass spectrometry using selected reaction monitoring that was conducted in an independent laboratory.

Development of potent and selective indomethacin analogues for the inhibition of AKR1C3 (Type 5 17ß-hydroxysteroid dehydrogenase/prostaglandin F synthase) in castrate-resistant prostate cancer.

Castrate-resistant prostate cancer (CRPC) is a fatal, metastatic form of prostate cancer. CRPC is characterized by reactivation of the androgen axis due to changes in androgen receptor signaling and/or adaptive intratumoral androgen biosynthesis. AKR1C3 is upregulated in CRPC where it catalyzes the formation of potent androgens. This makes AKR1C3 a target for the treatment of CRPC. AKR1C3 inhibitors should not inhibit AKR1C1/AKR1C2, which inactivate 5α-dihydrotestosterone. Indomethacin, used to inhibit cyclooxygenase, also inhibits AKR1C3 and displays selectivity over AKR1C1/AKR1C2. Parallel synthetic strategies were used to generate libraries of indomethacin analogues, which exhibit reduced cyclooxygenase inhibitory activity but retain AKR1C3 inhibitory potency and selectivity. The lead compounds inhibited AKR1C3 with nanomolar potency, displayed >100-fold selectivity over AKR1C1/AKR1C2, and blocked testosterone formation in LNCaP-AKR1C3 cells. The AKR1C3·NADP(+)·2'-des-methyl-indomethacin crystal structure was determined, and it revealed a unique inhibitor binding mode. The compounds reported are promising agents for the development of therapeutics for CRPC.

Development of potent and selective inhibitors of aldo-keto reductase 1C3 (type 5 17ß-hydroxysteroid dehydrogenase) based on N-phenyl-aminobenzoates and their structure-activity relationships.

Aldo-keto reductase 1C3 (AKR1C3; type 5 17ß-hydroxysteroid dehydrogenase) is overexpressed in castration resistant prostate cancer (CRPC) and is implicated in the intratumoral biosynthesis of testosterone and 5α-dihydrotestosterone. Selective AKR1C3 inhibitors are required because compounds should not inhibit the highly related AKR1C1 and AKR1C2 isoforms which are involved in the inactivation of 5α-dihydrotestosterone. NSAIDs, N-phenylanthranilates in particular, are potent but nonselective AKR1C3 inhibitors. Using flufenamic acid, 2-{[3-(trifluoromethyl)phenyl]amino}benzoic acid, as lead compound, five classes of structural analogues were synthesized and evaluated for AKR1C3 inhibitory potency and selectivity. Structure-activity relationship (SAR) studies revealed that a meta-carboxylic acid group relative to the amine conferred pronounced AKR1C3 selectivity without loss of potency, while electron withdrawing groups on the phenylamino B-ring were optimal for AKR1C3 inhibition. Lead compounds did not inhibit COX-1 or COX-2 but blocked the AKR1C3 mediated production of testosterone in LNCaP-AKR1C3 cells. These compounds offer promising leads toward new therapeutics for CRPC.

Overexpression of aldo-keto reductase 1C3 (AKR1C3) in LNCaP cells diverts androgen metabolism towards testosterone resulting in resistance to the 5α-reductase inhibitor finasteride.

Type 5 17ß-hydroxysteroid dehydrogenase (AKR1C3) is the major enzyme in the prostate that reduces 4-androstene-3,17-dione (Δ(4)-Adione) to the androgen receptor (AR) ligand testosterone. AKR1C3 is upregulated in prostate cancer (PCa) and castrate resistant prostate cancer (CRPC) that develops after androgen deprivation therapy. PCa and CRPC often depend on intratumoral androgen biosynthesis and upregulation of AKR1C3 could contribute to intracellular synthesis of AR ligands and stimulation of proliferation through AR signaling. To test this hypothesis, we developed an LNCaP prostate cancer cell line overexpressing AKR1C3 (LNCaP-AKR1C3) and compared its metabolic and proliferative responses to Δ(4)-Adione treatment with that of the parental, AKR1C3 negative LNCaP cells. In LNCaP and LNCaP-AKR1C3 cells, metabolism proceeded via 5α-reduction to form 5α-androstane-3,17-dione and then (epi)androsterone-3-glucuronide. LNCaP-AKR1C3 cells made significantly higher amounts of testosterone-17ß-glucuronide. When 5α-reductase was inhibited by finasteride, the production of testosterone-17ß-glucuronide was further elevated in LNCaP-AKR1C3 cells. When AKR1C3 activity was inhibited with indomethacin the production of testosterone-17ß-glucuronide was significantly decreased. Δ(4)-Adione treatment stimulated cell proliferation in both cell lines. Finasteride inhibited LNCaP cell proliferation, consistent with 5α-androstane-3,17-dione acting as the major metabolite that stimulates growth by binding to the mutated AR. However, LNCaP-AKR1C3 cells were resistant to the growth inhibitory properties of finasteride, consistent with the diversion of Δ(4)-Adione metabolism from 5α-reduced androgens to increased formation of testosterone. Indomethacin did not result in differences in Δ(4)-Adione induced proliferation since this treatment led to the same metabolic profile in LNCaP and LNCaP-AKR1C3 cells. We conclude that AKR1C3 overexpression diverts androgen metabolism to testosterone that results in proliferation in androgen sensitive prostate cancer. This effect is seen despite high levels of uridine glucuronosyl transferases suggesting that AKR1C3 activity can surmount the effects of this elimination pathway. Treatment options in prostate cancer that target 5α-reductase where AKR1C3 co-exists may be less effective due to the diversion of Δ(4)-Adione to testosterone.

Discovery of substituted 3-(phenylamino)benzoic acids as potent and selective inhibitors of type 5 17ß-hydroxysteroid dehydrogenase (AKR1C3).

Aldo-keto reductase 1C3 (AKR1C3) also known as type 5 17ß-hydroxysteroid dehydrogenase has been implicated as one of the key enzymes driving the elevated intratumoral androgen levels observed in castrate resistant prostate cancer (CRPC). AKR1C3 inhibition therefore presents a rational approach to managing CRPC. Inhibitors should be selective for AKR1C3 over other AKR1C enzymes involved in androgen metabolism. We have synthesized 2-, 3-, and 4-(phenylamino)benzoic acids and identified 3-(phenylamino)benzoic acids that have nanomolar affinity and exhibit over 200-fold selectivity for AKR1C3 versus other AKR1C isoforms. The AKR1C3 inhibitory potency of the 4'-substituted 3-(phenylamino)benzoic acids shows a linear correlation with both electronic effects of substituents and the pK(a) of the carboxylic acid and secondary amine groups, which are interdependent. These compounds may be useful in treatment and/or prevention of CRPC as well as understanding the role of AKR1C3 in endocrinology.

Inhibitors of type 5 17ß-hydroxysteroid dehydrogenase (AKR1C3): overview and structural insights.

There is considerable interest in the development of an inhibitor of aldo-keto reductase (AKR) 1C3 (type 5 17ß-hydroxysteroid dehydrogenase and prostaglandin F synthase) as a potential therapeutic for both hormone-dependent and hormone-independent cancers. AKR1C3 catalyzes the reduction of 4-androstene-3,17-dione to testosterone and estrone to 17ß-estradiol in target tissues, which will promote the proliferation of hormone dependent prostate and breast cancers, respectively. AKR1C3 also catalyzes the reduction of prostaglandin (PG) H(2) to PGF(2α) and PGD(2) to 9α,11ß-PGF(2), which will limit the formation of anti-proliferative prostaglandins, including 15-deoxy-Δ(12,14)-PGJ(2), and contribute to proliferative signaling. AKR1C3 is overexpressed in a wide variety of cancers, including breast and prostate cancer. An inhibitor of AKR1C3 should not inhibit the closely related isoforms AKR1C1 and AKR1C2, as they are involved in other key steroid hormone biotransformations in target tissues. Several structural leads have been explored as inhibitors of AKR1C3, including non-steroidal anti-inflammatory drugs, steroid hormone analogues, flavonoids, cyclopentanes, and benzodiazepines. Inspection of the available crystal structures of AKR1C3 with multiple ligands bound, along with the crystal structures of the other AKR1C isoforms, provides a structural basis for the rational design of isoform specific inhibitors of AKR1C3. We find that there are subpockets involved in ligand binding that are considerably different in AKR1C3 relative to the closely related AKR1C1 or AKR1C2 isoforms. These pockets can be used to further improve the binding affinity and selectivity of the currently available AKR1C3 inhibitors. Article from the special issue on Targeted Inhibitors.

Role of aldo-keto reductase enzymes in mediating the timing of parturition.

A better understanding of the mechanisms underlying parturition would provide an important step toward improving therapies for the prevention of preterm labor. Aldo-keto reductases (AKR) from the 1D, 1C, and 1B subfamilies likely contribute to determining the timing of parturition through metabolism of progesterone and prostaglandins. Placental AKR1D1 (human 5ß reductase) likely contributes to the maintenance of pregnancy through the formation of 5ß-dihydroprogesterone (DHP). AKR1C1, AKR1C2, and AKR1C3 catalyze the 20-ketosteroid and 3-ketosteroid reduction of progestins. They could therefore eliminate tocolytic progestins at term. Activation of the F prostanoid receptor by its ligands also plays a critical role in initiation of labor. AKR1C3 and AKR1B1 have prostaglandin (PG) F synthase activities that likely contribute to the initiation of labor. AKR1C3 converts PGH(2) to PGF(2α) and PGD(2) to 9α,11ß-PGF(2). AKR1B1 also reduces PGH(2) to PGF(2α), but does not form 9α,11ß-PGF(2). Consistent with the potential role for AKR1C3 in the initiation of parturition, indomethacin, which is a potent and isoform selective inhibitor of AKR1C3, has long been used for tocolysis.

Aldo-keto reductase 1C3 expression in MCF-7 cells reveals roles in steroid hormone and prostaglandin metabolism that may explain its over-expression in breast cancer.

Aldo-keto reductase (AKR) 1C3 (type 5 17beta-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD(2) to 9alpha,11beta-PGF(2), Delta(4)-androstenedione to testosterone, progesterone to 20alpha-hydroxyprogesterone, and to a lesser extent, estrone to 17beta-estradiol. We established MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other substrates at 0.1muM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17beta-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor alpha induced proliferation. MCF-7-AKR1C3 cells also reduced PGD(2), limiting its dehydration to form PGJ(2) products. The AKR1C3 product was confirmed as 9alpha,11beta-PGF(2) and quantified with a stereospecific stable isotope dilution liquid chromatography-mass spectrometry method. This method will allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD(2) on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor gamma (PPARgamma) signaling by reducing formation of 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)).

Steroid hormone transforming aldo-keto reductases and cancer.

Prostate and breast cancer are hormone-dependent malignancies of the aging male and female and require the local production of androgens and estrogens to stimulate cell proliferation. Aldo-keto reductases (AKR) play key roles in this process. In the prostate, AKR1C3 (type 5 17beta-HSD) reduces Delta(4)-androstene-3,17-dione to yield testosterone while AKR1C2 (type 3 3alpha-HSD) eliminates 5alpha-dihydrotestosterone (5alpha-DHT), and AKR1C1 forms 3beta-androstanediol (a ligand for ERbeta). In the breast, AKR1C3 forms testosterone, which is converted to 17beta-estradiol by aromatase or reduces estrone to 17beta-estradiol directly. AKR1C3 also acts as a prostaglandin (PG) F synthase and forms PGF(2alpha) and 11beta-PGF(2alpha), which stimulate the FP receptor and prevent the activation of PPARgamma by PGJ(2) ligands. This proproliferative signaling may stimulate the growth of hormone-dependent and -independent prostate and breast cancer.

Type 5 17beta-hydroxysteroid dehydrogenase/prostaglandin F synthase (AKR1C3): role in breast cancer and inhibition by non-steroidal anti-inflammatory drug analogs.

Aldo-keto reductase (AKR) 1C3 catalyzes the NADPH-dependent reduction of Delta(4)-androstene-3,17-dione to yield testosterone, reduction of estrone to yield 17beta-estradiol and reduction of progesterone to yield 20alpha-hydroxyprogesterone. In addition, it functions as a prostaglandin (PG) F synthase and reduces PGH(2) to PGF(2)alpha and PGD(2) to 11beta-PGF(2). Immunohistochemistry showed that AKR1C3 is over-expressed in invasive ductal carcinoma of the breast. Retroviral expression of AKR1C3 in MCF-7 breast carcinoma cells shows that each of the assigned reactions occur in a breast cell microenvironment. Steroid and prostaglandin conversions were monitored by radiochromatography. Prostaglandin conversion was validated by a second method using HPLC coupled to APCI-MRM/MS. The combined effect of the AKR1C3 catalyzed 17- and 20-ketosteroid reductions will be to increase the 17beta-estradiol:progesterone ratio in the breast. In addition, formation of PGF(2) epimers would activate F prostanoid receptors and deprive PPARgamma of its putative anti-proliferative PGJ(2) ligands. Thus, AKR1C3 is a source of proliferative signals and a potential therapeutic target for hormone-dependent and -independent breast cancer. Two strategies for AKR1C3 inhibition based on non-steroidal anti-inflammatory drugs were developed. The first strategy uses the Ullmann coupling reaction to generate N-phenylanthranilate derivatives that inhibit AKR1C enzymes without affecting PGH(2) synthase (PGHS) 1 or PGHS-2. The second strategy exploits the selective inhibition of AKR1C3 by indomethacin, which did not inhibit highly related AKR1C1 or AKR1C2. Using known structure-activity relationships for the inhibition of PGHS-1 and PGHS-2 by indole acetic acids we obtained N-(4-chlorobenzoyl)-melatonin as a specific AKR1C3 inhibitor (K(I)=6.0muM) that does not inhibit PGHS-1, PGHS-2, AKR1C1, or AKR1C2. Both strategies are informed by crystal structures of ternary AKR1C3.NADP(+).NSAID complexes. The identification of NSAID analogs as specific inhibitors of AKR1C3 will help validate its role in the proliferation of breast cancer cells.

An indomethacin analogue, N-(4-chlorobenzoyl)-melatonin, is a selective inhibitor of aldo-keto reductase 1C3 (type 2 3alpha-HSD, type 5 17beta-HSD, and prostaglandin F synthase), a potential target for the treatment of hormone dependent and hormone independent malignancies.

Aldo-keto reductase (AKR) 1C3 (type 2 3alpha-HSD, type 5 17beta-HSD, and prostaglandin F synthase) regulates ligand access to steroid hormone and prostaglandin receptors and may stimulate proliferation of prostate and breast cancer cells. NSAIDs are known inhibitors of AKR1C enzymes. An NSAID analogue that inhibits AKR1C3 but is inactive against the cyclooxygenases and the other AKR1C family members would provide an important tool to examine the role of AKR1C3 in proliferative signaling. We tested NSAIDs and NSAID analogues for inhibition of the reduction of 9,10-phenanthrenequinone (PQ) catalyzed by AKR1C3 and the closely related isoforms AKR1C1 and AKR1C2. Two of the compounds initially screened, indomethacin and its methyl ester, were specific for AKR1C3 versus the other AKR1C isoforms. Based on these results and the crystal structure of AKR1C3, we predicted that N-(4-chlorobenzoyl)-melatonin (CBM), an indomethacin analogue that does not inhibit the cyclooxygenases, would selectively inhibit AKR1C3. CBM inhibited the reduction of PQ by AKR1C3, but did not significantly inhibit AKR1C1 or AKR1C2. Indomethacin and CBM also inhibited the AKR1C3-catalyzed reduction of Delta(4)-androstene-3,17-dione but did not significantly inhibit the reduction of steroid hormones catalyzed by AKR1C1 or AKR1C2. The pattern of inhibition of AKR1C3 by indomethacin and CBM was uncompetitive versus PQ, but competitive versus Delta(4)-androstene-3,17-dione, indicating that two different inhibitory complexes form during the ordered bi bi reactions. The identification of CBM as a specific inhibitor of AKR1C3 will aid the investigation of its roles in steroid hormone and prostaglandin signaling and the resultant effects on cancer development.

Formation of 1,4-dioxo-2-butene-derived adducts of 2'-deoxyadenosine and 2'-deoxycytidine in oxidized DNA.

Oxidation of deoxyribose in DNA produces a variety of electrophilic residues that are capable of reacting with nucleobases to form adducts such as M(1)dG, the pyrimidopurinone adduct of dG. We now report that deoxyribose oxidation in DNA leads to the formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA. We previously demonstrated that these adducts arise in reactions of nucleosides and DNA with trans-1,4-dioxo-2-butene, the beta-elimination product of the 2-phosphoryl-1,4-dioxobutane residue arising from 5'-oxidation of deoxyribose in DNA, and with cis-1,4-dioxo-2-butene, a metabolite of furan. Treatment of DNA with enediyne antibiotics capable of oxidizing the 5'-position of deoxyribose (calicheamicin and neocarzinostatin) led to a concentration-dependent formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA, while the antibiotic bleomycin, which is capable of performing only 4-oxidation of deoxyribose, did not give rise to the adducts. The nonspecific DNA oxidant, gamma-radiation, also produced the adducts that represented approximately 0.1% of the 2-phosphoryl-1,4-dioxobutane residues formed during the irradiation. These results suggest that the oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA could represent endogenous DNA lesions arising from oxidative stresses that also give rise to other DNA adducts.

Detection of DNA adducts derived from the reactive metabolite of furan, cis-2-butene-1,4-dial.

Furan is a toxic and carcinogenic compound used in industry and commonly found in the environment. The mechanism of furan's carcinogenesis is not well-understood and may involve both genotoxic and nongenotoxic pathways. Furan undergoes oxidation by cytochrome P450 to cis-2-butene-1,4-dial, which is thought to mediate furan's toxic effects. Consistently, cis-2-butene-1,4-dial readily reacts with glutathione, amino acids, and nucleosides. To determine the importance of DNA alkylation in furan-induced carcinogenesis, we developed an assay for the detection of cis-2-butene-1,4-dial-derived DNA adducts. DNA samples were treated with O-benzyl-hydroxylamine, which reacts with the aldehyde functionality of the DNA adducts. Enzyme hydrolysates of these samples were then analyzed by capillary electrospray tandem mass spectrometry with selected reaction monitoring. The dCyd and dAdo adducts were detected in digests of DNA treated with nanomolar concentrations of cis-2-butene-1,4-dial. In addition, these adducts were present in DNA isolated from Ames assay strain TA104 treated with mutagenic concentrations of cis-2-butene-1,4-dial. These data support the hypothesis that cis-butene-1,4-dial is a genotoxic metabolite of furan. This method will allow us to explore the role of these adducts in furan-induced carcinogenesis.

The formation of substituted 1,N6-etheno-2'-deoxyadenosine and 1,N2-etheno-2'-deoxyguanosine adducts by cis-2-butene-1,4-dial, a reactive metabolite of furan.

Furan is an environmental chemical that induces liver toxicity and tumor formation in rodents, leading to its classification as a probable human carcinogen. cis-2-Butene-1,4-dial, the metabolite considered responsible for furan's toxicological effects, is mutagenic in the Ames assay and reacts with 2'-deoxycytidine (dCyd), 2'-deoxyadenosine (dAdo), and 2'-deoxyguanosine (dGuo) to form previously characterized diastereomeric adducts. The initially formed dCyd adducts are stable to rearrangement, while the dAdo and dGuo adducts are unstable and rearrange to form secondary products. On the basis of UV absorbance, fluorescence, 1H NMR, and mass spectral data, the rearrangement product of the dAdo adduct was identified as the substituted etheno-dAdo adduct, 1''-[3-(2'-deoxy-beta-D-erythropentafuranosyl)-3H-imidazo[2,1-i]purin-8-yl]ethane-2''-al. The NMR characterization of the O-methyloxime derivative of the secondary dGuo adduct, along with mass spectral and UV data on the underivatized adduct, allowed for its structural assignment as the substituted etheno-dGuo compound, 3-(2'-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(ethane-2''-al)[1,2-alpha]purine-9-one. The characterization of the primary and secondary products formed in the reaction of cis-2-butene-1,4-dial with nucleosides is important for understanding the mechanism of furan-induced carcinogenesis. These secondary adducts retain a reactive aldehyde with the potential to form cross-links and are likely to contribute significantly to furan's toxic and carcinogenic effects.

Characterization of nucleoside adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan.

Furan is a hepatic toxicant and carcinogen in rodents. Its microsomal metabolite, cis-2-butene-1,4-dial, is mutagenic in the Ames assay. Consistent with this observation, cis-2-butene-1,4-dial reacts with 2'-deoxycytidine, 2'-deoxyguanosine, and 2'-deoxyadenosine to form diastereomeric adducts. HPLC analysis indicated that the rate of reaction with deoxyribonucleosides was dependent on pH. At pH 6.5, the relative reactivity was 2'-deoxycytidine > 2'-deoxyguanosine > 2'-deoxyadenosine whereas it was 2'-deoxyguanosine > 2'-deoxycytidine > 2'-deoxyadenosine at pH 8.0. Thymidine did not react with cis-2-butene-1,4-dial. The primary 2'-deoxyguanosine and 2'-deoxyadenosine reaction products were unstable and decomposed to secondary products. NMR and mass spectral analysis indicated that the initial 2'-deoxyadenosine and 2'-deoxyguanosine reaction products were hemiacetal forms of 3-(2'-deoxy-beta-D-erthyropentafuranosyl)-3,5,6,7-tetrahydro-6-hydroxy-7-(ethane-2''-al)-9H-imidazo[1,2-alpha]purine-9-one (structure 2) and 3-(2'-deoxy-beta-D-erythropentafuranosyl)-3,6,7,8-tetrahydro-7-(ethane-2''-al)-8-hydroxy-3H-imidazo[2,1-i]purine (structure 4), respectively. These adducts resulted from the addition of cis-2-butene-1,4-dial to the exo- and endocyclic nitrogens of 2'-deoxyadenosine and 2'-deoxyguanosine. The data provide support for the hypothesis that cis-2-butene-1,4-dial is an important genotoxic intermediate in furan-induced carcinogenesis.