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The biotransformation and excretion of darolutamide were investigated in a phase I study. Six healthy male volunteers received a single dose of 300 mg 14C-darolutamide as an oral solution in the fasted state. Plasma, urine, and feces samples were analyzed for mass balance evaluation by liquid scintillation counting (LSC). Metabolite profiling and identification were determined using liquid chromatography mass-spectrometry with off-line radioactivity detection using LSC. Complete mass balance was achieved, with mean radioactivity recovery of 95.9% within 168 hours (63.4% in urine, 32.4% in feces). The administered 1:1 ratio of (S,R)- and (S,S)-darolutamide changed to approximately 1:5, respectively, in plasma. Darolutamide and the oxidation product, keto-darolutamide, were the only components quantifiable by LSC in plasma, accounting for 87.4% of total radioactivity, with a 2.1-fold higher plasma exposure for keto-darolutamide. Aside from darolutamide, the most prominent metabolites in urine were O-glucoronide (M-7a/b) and N-glucuronide (M-15a/b), as well as pyrazole sulfates (M-29, M-24) and glucuronides (M-21, M-22) resulting from oxidative cleavage of the parent. The darolutamide diastereomers were mainly detected in feces. In vitro assays showed that darolutamide metabolism involves a complex interplay between oxidation and reduction, as well as glucuronidation. Interconversion of the diastereomers involves oxidation to keto-darolutamide, primarily mediated by CYP3A4, followed by reduction predominantly catalyzed by cytosolic reductase(s), with aldo-keto reductase 1C3 playing the major role. The latter reaction showed stereoselectivity with preferential formation of (S,S)-darolutamide. SIGNIFICANCE STATEMENT: The metabolism and excretion of darolutamide in humans revealed that oxidation (CYP3A4) and glucuronidation (UGT1A9, UGT1A1) were the main metabolic routes of elimination. Direct excretion also contributed to overall clearance. The two pharmacologically equipotent diastereomers of darolutamide interconvert primarily via oxidation to the active metabolite keto-darolutamide, followed by reduction predominantly by cytosolic reductase(s). The latter reaction showed stereoselectivity with preferential formation of (S,S)-darolutamide. Data indicate a low drug-drug interaction potential of darolutamide with inducers or inhibitors of metabolizing enzymes.

Background: Saini et al. recently investigated the pharmacokinetics of darolutamide and its diastereomers in vitro and in vivo in Balb/c mice, reporting higher levels of (S,S)-darolutamide than (S,R)-darolutamide following intravenous or oral dosing, and interconversion of (S,R)-darolutamide to (S,S)-darolutamide. Objective: To present our in vitro and in vivo studies of darolutamide pharmacokinetics in mice, which contrast with the findings of Saini et al. Methods: Nude male Balb/c mice were orally dosed for 7 days with 25, 50, or 100 mg/kg of darolutamide twice daily. Pharmacokinetic parameters in plasma and tissue samples were assessed by liquid chromatography-tandem mass spectrometry. Metabolism and interconversion of darolutamide and its diastereomers were investigated in cryopreserved Balb/c mouse hepatocytes. Protein binding was determined in plasma samples by equilibrium dialysis. Results : On day 7, Cmaxwas reached 30 min after the last dose. Rapid formation and greater exposure of keto-darolutamide versus darolutamide were observed. Plasma exposure of (S,R)-darolutamide was 3-5-fold higher than that of (S,S)-darolutamide. The fraction of unbound keto-darolutamide was almost 6-fold lower than for darolutamide. Conclusion: The darolutamide diastereomer ratio changes upon administration in mice and other species due to interconversion through keto-darolutamide. This is not considered clinically relevant since both diastereomers and keto- darolutamide are pharmacologically similar in vitro. Based on the high protein binding of keto-darolutamide, its contribution in vivo in humans is considered low.

1. Darolutamide is a novel selective androgen receptor antagonist consisting of two pharmacologically equipotent diastereoisomers. The absorption, distribution, metabolism and excretion properties of darolutamide in rats are reported.2. Non- or [

Objective To investigate the pharmacokinetics and excretion balance of [ 14 C]-OR-1896, a pharmacologically active metabolite of levosimendan, in six healthy male subjects. In addition, pharmacokinetic parameters of total radiocarbon and the deacetylated congener, OR-1855, were determined. Methods OR-1896 was administered as a single intravenous infusion of 200 μg of [ 14 C]-OR-1896 (specific activity 8.6 MBq/mg) over 10 min. The pharmacokinetic parameters were calculated by three-compartmental methods. Results During the 14-day collection of urine and faeces, excretion (±S.D.) averaged 94.2 ± 1.4% of the [ 14 C]-OR-1896 dose. Mean recovery of radiocarbon in urine was 86.8 ± 1.9% and in faeces 7.4 ± 1.5%. Mean terminal elimination half-life of OR-1896 ( t 1/2 ) was 70.0 ± 44.9 h. Maximum concentrations of OR-1855 were approximately 30% to that of OR-1896. Total clearance and the volume of distribution of OR-1896 were 2.0 ± 0.4 l/h and 175.6 ± 74.5 l, respectively. Renal clearances of OR-1896 and OR-1855 were 0.9 ± 0.4 l/h and (5.4 ± 2.3) × 10 −4 l/h, respectively. Conclusions This study provides data to demonstrate that nearly one half of OR-1896 is eliminated unchanged into urine and that the active metabolites metabolite of levosimendan remain in the body longer than levosimendan. The remaining half of OR-1896 dose is eliminated through other metabolic routes, partially through interconversion back to OR-1855 with further metabolism of OR-1855. Given the fact that the pharmacological activity and potency of OR-1896 is similar to levosimendan, these results emphasize the clinical significance of OR-1896 and its contribution to the long-lasting effects of levosimendan.

Nicotine biotransformation affects the smoking habits of addicted individuals and therefore their health risk. Using an improved analytical method, we have discovered that the human UDP-glucuronosyltransferase (UGT) 2B10, a liver enzyme previously unknown to conjugate nicotine or exhibit considerable activity toward any compound, plays a major role in nicotine inactivation by direct conjugation with glucuronic acid at the aromatic nitrogen atom. The K(m) value of recombinant UGT2B10 for nicotine (0.29 mM) was similar to that determined for human liver microsomes (0.33 mM), whereas the K(m) value of UGT1A4 for nicotine was almost 10-fold greater (2.4 mM). UGT2B10 was also more active than UGT1A4 in N-glucuronidation of cotinine (oxidative nicotine metabolite), whereas UGT2B7 exhibited only low nicotine glucuronidation activity and was essentially inactive toward cotinine. UGT1A9 did not glucuronidate nicotine or cotinine. Quantitative reverse transcription-polymerase chain reaction showed that UGT2B10 mRNA was exclusively expressed in human liver, whereas UGTs 1A4 and 2B7 were expressed at comparable, although somewhat lower, levels in liver and several other extrahepatic tissues, including kidney and intestine. These findings for UGT2B10 (but not for UGT1A4 and UGT2B7) were mirrored by human tissue activities because nicotine and cotinine glucuronidation rates in intestine microsomes were less than 0.1% that of human liver microsomes. These novel findings solve two seemingly separate questions: which UGT is primarily responsible for nicotine glucuronidation in human liver, and what conjugation reactions are catalyzed by UGT2B10.

This study describes urinary excretion of two nucleobase adducts derived from styrene 7,8-oxide (SO), i.e., 7-(2-hydroxy-1-phenylethyl)guanine (N7alphaG) and 7-(2-hydroxy-2-phenylethyl)guanine (N7betaG), as well as a formation of N7-SO-guanine adducts in lungs and liver of two month old male NMRI mice exposed to styrene by inhalation in a 3-week subacute study. Strikingly higher excretion of both isomeric nucleobase adducts in the first day of exposure was recorded, while the daily excretion of nucleobase adducts in following time intervals reached the steady-state level at 4.32+1.14 and 6.91+1.17 pmol/animal for lower and higher styrene exposure, respectively. beta-SO-guanine DNA adducts in lungs increased with exposure in a linear way (F=13.7 for linearity and 0.17 for non-linearity, respectively), reaching at the 21st day the level of 23.0 adducts/10(8) normal nucleotides, i.e., 0.74 fmol/microg DNA of 7-alkylguanine DNA adducts for the concentration of 1500 mg/m3, while no 7-SO-guanine DNA adducts were detected in the liver after 21 days of inhalation exposure to both of styrene concentrations. A comparison of 7-alkylguanines excreted in urine with 7-SO-guanines in lungs (after correction for depurination and for missing alpha-isomers) revealed that persisting 7-SO-guanine DNA adducts in lungs account for about 0.5% of the total alkylation at N7 of guanine. The total styrene-specific 7-guanine alkylation accounts for about 1.0x10(-5)% of the total styrene uptake, while N1-adenine alkylation contributes to this percentage only negligibly.

This report reviews styrene biotransformation, including minor metabolic routes, and relates metabolism to the genotoxic effects and possible styrene-related carcinogenicity. Styrene is shown to require metabolic activation in order to become notably genotoxic and styrene 7,8-oxide is shown to contribute quantitatively by far the most (in humans more than 95%) to the genotoxicity of styrene, while minor ring oxidation products are also shown to contribute to local toxicities, especially in the respiratory system. Individual susceptibility depending on metabolism polymorphisms and individual DNA repair capacity as well as the dependence of the nonlinearity of the dose-response relationships in the species in question and the consequences for risk evaluation are analyzd.

We evaluated our data on the occupational exposure to styrene in lamination workers. The battery of parameters included markers of external and internal exposure and biomarkers of biological effects and susceptibility. DNA repair capacities have been determined in both exposed and control groups. Styrene workplace concentration significantly correlated with styrene concentration in blood, exhaled air and urinary mandelic acid. Haemoglobin and O 6 -styrene oxide (SO)-guanine DNA adducts were significantly higher in exposed subjects as compared to controls and correlated with exposure parameters. In styrene-exposed workers 1-SO-adenine DNA adducts were detected (2.6 per 10 9 dNp), while in controls these adducts were below the detection limit. 1-SO-adenine adduct levels were affected by both acute and cumulative exposure ( P =0.001, F =86.0 and P =0.017, F =59.0, respectively) and associated with cytochrome P450 2E1 (CYP2E1) polymorphisms ( R 2 =0.442). Mutant frequencies (MF) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus appeared to accumulate with exposure over time and were associated with glutathione S -transferase P1 (GSTP1) polymorphism. DNA repair capacity increased with the exposure, except for the group exposed to the highest styrene concentration. In this particular group, increased DNA repair capacity to remove oxidative DNA damage was found.

Objective. Based on our previous studies we analysed DNA and haemoglobin adducts, single-strand breaks in DNA, chromosomal aberrations and HPRT mutant frequencies in styrene-exposed workers, in relation to employment time, with respect to markers of individual susceptibility. In our study the risk assessment of genotoxic styrene in occupationally exposed humans is reviewed in the light of adaptation and/or population selection. Various styrene-induced DNA adducts (representing important integral measures of DNA damage) are discussed as potential powerful biomarkers of styrene exposure, with respect to DNA repair. Methods. The complexity of followed markers required multiple methodological approaches, including modified 32P-post-labelling assay, PCR-based methods for genotyping, cytogenetic analysis, T-cell cloning assay, comet assay and tests for DNA repair capacity, and statistical tests. Results. Haemoglobin and DNA adducts, single-strand breaks in DNA, chromosomal aberrations and mutant frequencies at the HPRT gene were assessed with respect to the duration of exposure. No apparent accumulation of these biomarkers was found to be dependent on the years of employment, suggesting that adaptive processes play a role in chronically exposed workers. Statistically significant differences between the exposed and control individuals were found for the deduced epoxide hydrolase activity (P=0.041) and the frequency of heterozygous Ala/Val genotype in GSTP1, exon 5 (P=0.025). Significantly increased DNA repair capacity was found in styrene-exposed laminators compared with control individuals. Conclusions. In the light of present knowledge well-designed population studies on styrene genotoxicity are needed. Such studies should cover important areas of individual susceptibility (metabolising and repair enzymes, distributions of genetic polymorphisms) and possible role of adaptation, as well as the crucial role of DNA repair. Additionally, genotoxic effects of in-vivo formed 3,4-styrene oxide should be also addressed.

The possible carcinogenicity of styrene is believed to be related to the DNA-binding properties of styrene 7,8-oxide (SO). In order to compare the intrinsic reactivity of the different nucleophilic sites in DNA towards SO and to evaluate the candidates for human biomonitoring we have determined the second-order rate constants and stabilities of several SO-adducts in double-stranded DNA. These include alpha- and beta-isomers of N7-substituted and alphaN(2)-substituted guanines, alpha- and betaN3-substituted and alphaN(6)-substituted adenines as well as betaN3- and alphaN(4)-substituted cytosines. The highest rate constants were found for the spontaneously depurinating N7-guanines being ca. 3-15-fold higher than those for the stable adducts. When the relative proportions of different alkylation products were determined in course of time, after a single addition of SO, the labile N7-guanines and N3-adenines were the major products at early time points. After 144 h of incubation at 37 degrees C, alphaN(6)-SO-adenine and alphaN(2)-SO-guanine as well as betaN3-SO-uracil were the major adducts. Regarding human biomonitoring, the N7-substituted guanines should be one of the main targets because of the high reactivity of the N7-atom of guanine. However, in the case of chronic styrene exposures the chemically more stable DNA adducts may become important.

Genotoxic and clastogenic effects of styrene were studied in mice. Male NMRI mice were exposed by inhalation to styrene in concentrations of 750 and 1500 mg/m3 for 21, 7, 3 and 1 days (6 h/day, 7 days/week). Followed parameters included styrene in blood, specific styrene oxide (SO) induced DNA adducts, DNA strand breaks and micronuclei. The formation of SO induced 7-SO-guanines and 1-SO-adenines in DNA was analysed from lung tissues by two versions of the 32P-postlabeling technique. In lungs after 21 days of exposure to 1500 mg/m3 the level of 7-SO-guanine was 23.0+/-11.9 adducts/10(8) normal nucleotides, while 1-SO-adenine was detected at the levels of 0.6+/-0.2 adducts/10(8) normal nucleotides. Both 7-SO-guanines and 1-SO-adenines strongly correlated with exposure parameters, particularly with styrene concentration in blood (r=0.875, P=0.0002 and r=0.793, P=0.002, respectively). DNA breaks were measured in peripheral lymphocytes, bone marrow cells and liver cells using comet assay. To discern oxidative damage and abasic sites, endonuclease III was used. In bone marrow of exposed mice slight increase of strand breaks can be detected after 7 days of inhalation. A significant increase was revealed in the endonuclease III-sensitive sites after 21 days of inhalation in bone marrow. In the liver cells inhalation exposure to both concentrations of styrene did not virtually affect either levels of DNA single-strand breaks or endonuclease III-sensitive sites. The inhalation of 1500 mg/m3 of styrene induced significant increase of micronuclei after 7 days of exposure (10.4+/-2.5/1000 cells, i.e. twice higher micronuclei frequency than in controls). After 21 days of inhalation no significant difference between the control group and the two exposed groups was observed. Whether the decrease of micronuclei after 21 days of inhalation was due to the inhibition of cell proliferation caused by styrene or due to the natural elimination of chromatide fragments, remains to be clarified. An interesting link has been found between DNA single-strand breaks in bone marrow and frequencies of micronuclei (r=0.721, P=0.028).

Drug metabolism can result in the formation of highly reactive metabolites that are known to play a role in toxicity resulting in a significant proportion of attrition during drug development and clinical use. Thus, the earlier such reactivity was detected, the better. This review summarizes our multi-year project, together with pertinent literature, to examine a battery of in vitro tests capable of detecting the formation of reactive metabolites. Principal prerequisites for such tests were delineated: chemicals known/not known to cause tissue injury and produce reactive metabolites, activation system (mainly human-derived), small- and large-molecular targets (small-molecular trappers, peptides, proteins), analytical techniques (mass spectrometry), and cellular toxicity biomarkers. The current status of in vitro tools to detect reactive intermediates is the following: 1. Small-molecular trapping agents such glutathione or cyanide detect the production of reactive species with high sensitivity by proper MS technique. However, it seems that also putative “negatives” give rise to corresponding adducts. 2. Results from peptide and dG (DNA targeting) trapper studies are generally in line with those of small-molecular trappers, although also important differences exist. These two trapping platforms do not overlap. 3. It is anticipated that the in vitro adduct studies could be fully interpreted only in conjunction with toxicity biomarker (such as the Nrf2 pathway) information from whole cells or tissues. However, while there are tools to characterize the chemical liability and there are correlation between individual/integrated endpoints and toxicity, there are still severe gaps in understanding the mechanisms behind the link between reactive metabolites and adverse effects.

The reaction between 2'-deoxycytidine and styrene 7,8-oxide (SO) resulted in alkylation at the 3-position and at the O(2)-position through the alpha- and beta-carbons of the epoxide but at the N(4)-position only through the alpha-carbon. The 3-alkylated adducts were found to deaminate to the corresponding 2'-deoxyuridine adducts (37 degrees C, pH 7.4) with half-lives of 6 min and 2.4 h for the alpha- and beta-isomers, respectively. The N(4)-alkylated products were stable at neutral pH. The O(2)-alkylated products were unstable being prone to depyrimidation and to isomerisation between alpha- and beta-isomers. In SO-treated double-stranded DNA, enzymatic hydrolysis allowed the identification of the beta3-deoxyuridine and alphaN(4)-deoxycytidine adducts (1.9 and 0.5% of total alkylation, respectively), in addition to the previously identified DNA-adducts. The 3-substituted uracil may have implications for the mutagenicity of SO.

FC1271a is a novel triphenylethylene compound with a tissue-selective profile of estrogen agonistic and weak antagonistic effects. It specifically binds to the estrogen receptor alpha and beta with affinity closely similar to that of toremifene and tamoxifen. To study the in vivo effects of the compound, 4-month-old rats were sham operated (sham) or ovariectomized (OVX) and treated daily for 4 weeks with various doses of FC1271a or vehicle (orally). FC1271a was able to oppose OVX-induced bone loss by maintaining the trabecular bone volume of the distal femur. Accordingly, the OVX-induced loss of bone strength was prevented at doses of 1 and 10 mg/kg. FC1271a also prevented the OVX-induced increase in serum cholesterol in a dose-dependent manner. No significant changes in uterine wet weight or morphology were observed in the OVX-rats treated with 0.1 or 1 mg/kg FC1271a, but at a dose of 10 mg/kg it had a slightly estrogenic effect. In immature rats the effect of FC1271a on uterine wet weight was less stimulatory than that of toremifene or tamoxifen, but more stimulatory than that of raloxifene or droloxifene. The appearance of the dimethylbenzanthracene (DMBA)-induced mammary tumors was inhibited by treatment of DMBA-treated rats with FC1271a in a dose-dependent manner. In human MCF-7 breast cancer cell tumors raised in nude mice in the presence of estrogen, the growth and expression of pS2 marker gene could not be maintained after estrogen withdrawal by treatment with FC1271a. No formation of DNA adducts was observed in the liver of the FC1271a-treated rats. In conclusion, the bone-sparing, antitumor, and cholesterol-lowering effects of FC1271a combined with a low uterotropic activity and lack of liver toxicity indicate that FC1271a could be an important alternative in planning antiosteoporosis therapy for estrogen deficiency.

Styrene 7,8-oxide (SO), a major metabolite of styrene, is classified as a probable human carcinogen. In the present work, salmon testis DNA was reacted with SO and the alkylation products were analysed after sequential depurination in neutral or acidic conditions followed by HPLC separation and UV-detection. A novel finding was that the N-3 position of adenine was the next most reactive alkylation site in double-stranded DNA, comprising 4% of the total alkylation, as compared to alkylation at the N-7 position of guanine, 93% of the total alkylation. Both alpha- and beta-products of SO were formed at these two sites. Other modified sites were N2-guanine (1.5%, alpha-isomer), 1-adenine (0.4%, both isomers) and N6-adenine (0.7%, both isomers) as well as 1-hypoxanthine (0.1%, alpha-isomer), formed by deamination of the corresponding 1-adenine adduct. The results indicated that in double-stranded DNA N-7 of guanine and N-3 of adenine account for 97% of alkylation by SO. However, these abundant adducts are not stable, the half-life of depurination in DNA for 3-substituted adenines being approximately 10 and approximately 20 h, for alpha- and beta-isomers, respectively, and 51 h for both isomers of 7-substituted guanines.

Styrene 7,8-oxide (SO), a reactive epoxide that has been classified as a probable human carcinogen, was allowed to react under alkaline conditions with 2′-deoxyguanosine and thymidine monophosphates as well as with the dinucleotide dGpdT. The reaction products were separated by high-performance liquid chromatography and were characterised by UV and electrospray mass spectroscopy, the latter showing the ability to differentiate the isomerism of the hydroxyphenylethyl moiety of the adduct. The main alkylation products of the deoxyguanosine monophosphates at high pH were those reacted at the 1- (through the β-carbon of the epoxide), N2- (α-carbon) and 7-positions (β- and α-carbons) of guanine followed by alkylation of the phosphate group. The formation of a novel diastereomeric pair of N 2-guanine adducts connected to the β-carbon of SO was identified and characterised. Furthermore, two different geometrical isomers of 1-guanine were detected. For thymidine nucleotide, base alkylation under neutral conditions was almost negligible, but at pH 10.5, alkylation at the 3-position was very prominent. The same base-adducts and the pH-effects were observed in the case of dGpdT alkylation. However, no phosphate alkylation was detected in the case of the dinucleotide.

The structures and protonation energies of aminoguanidine and aminoguanidinium ion were obtained from quantum chemical calculations by using the density functional method B3-LYP and the standard basis set 6 -31 G(d ). The energy differences between the various forms of the two possible tautomers, the endiamine and the imidamide form, were investigated. The endiamine was found to be more stable than the most stable form of the imidamide by 5 kcal/mol in the gas phase. The proton affinity of am inoguanidine was found to be 241.4 kcal/mol (B3 -LYP/6 -31 G(d )) in the gas phase. The liquidation pKa2 for amininoguanidine and the guanidinium ion was determined to be 11.5 ± 0.1. The reference values for guanidine free base and the guanidinium ion are 235.7 kcal/mol and 13.6, respectively. The difference in the order of basicity in the gas phase and in aqueous solution between aminoguanidine and guanidine is discussed in terms of symmetry, resonance, delocalisation, rehybridisation of nitrogen atoms, and electrostatic repulsion.

DNA isolated from livers of rats receiving tamoxifen was analysed by the 32P-postlabelling method. The postlabelled DNA hydrolysis mixture was analysed both by reversed-phase HPLC with 32P on-line detection and by TLC on polyethyleneimine plates followed by autoradiography. Using the HPLC method, five well separated adduct peaks could be detected, while by the TLC method, two groups of adduct spots were observed. The detection limit of the TLC assay was lower (0.5 adducts/10(10) nucleotides) than that of the HPLC assay (3 adducts/10(10) nucleotides). Thus, the TLC assay is more sensitive but also more laborious. The advantages of the HPLC assay were, in addition to better resolution, the ease of quantification and operation.

The crystal and molecular structure of aminoguanidine hemioxalate, a salt in which aminoguanidine exists in the monocation form, was determined by single crystal X-ray diffraction. The salt crystallizes in the monoclinic space group P2(1)/n with unit cell dimensions ofa=4.95,b=10.46,c=10.40 A, β=92.57°, andZ=4. The structure contains one oxalate ion for every two CN4H 7 + ions, the latter being practically planar. The structure of the monocation is largely similar to those of aminoguanidine dications except that the monocation is devoid of one of the protons attached to the terminal hydrazine nitrogen. This result is of interest considering the synthesis of amidinohydrazones, indicating that the concentration of the active nucleophile is nearly maximal even when aminoguanidine exists in the monocation form. Therefore, the synthesis of amidinohydrazones should be performed in thepH range in which aminoguanidine exists mainly in the monocation form,i.e. at apH higher than 2. There is, however, no need to elevate thepH to values at which a considerable proportion of aminoguanidine exists as the free base.

Butadiene monoepoxide (BMO), epoxybutanediol (EBD) and diepoxybutane (DEB) are reactive metabolites of 1,3-butadiene (BD), an important industrial chemical classified as a probable human carcinogen. The covalent interactions of these metabolites with DNA lead to the formation of DNA adducts which may induce mutations or other types of DNA damage, resulting in tumour formation. In the present study, two pairs of diastereomeric N-1-BMO-adenine adducts were identified in the reaction of BMO with 2´-deoxyadenosine-5´-monophosphate (5´-dAMP). The major products formed by reacting EBD with 2´-deoxyguanosine-5´-monophosphate (5´-dGMP) were characterized as diastereomeric N-7-(2´,3´,4´-trihydroxybut-1´-yl)-5´-dGMP by UV and electrospray mass spectrometry. The formation of N-7-BMO-guanine adducts (1´-carbon, 60; 2´carbon, 54/10(4) nucleotides) in BMO-treated DNA was about four times higher than that of N-1-BMO-adenine adducts (1´-carbon, 20; 2´-carbon, 8.7/10(4) nucleotides). However, the recovery of N-1-BMO-adenine adducts in DNA (45 ± 5%) was two times higher than that of N-7-guanine adducts (20 ± 4%) by 32P-postlabelling analysis. Using the 32P-postlabelling/ HPLC assay, N-1-BMO-adenine, N-7-BMO-guanine and N-7-EBDguanine adducts were detected in BMO- or DEB-treated DNA and in liver DNA of rats exposed to BD by inhalation. The amount of N-7-EBD-guanine adducts (11/10(8) nucleotides) in rat liver was about three-fold higher than N-7-BMO-guanine adducts (4.0/10(8) nucleotides). The novel finding of N-1-BMO-adenine adducts formed in vivo may contribute to the understanding of the mechanisms of BD carcinogenic action.

Styrene oxide (SO), a reactive metabolite of styrene, modifies DNA at several nucleophilic sites. In the present work we have determined the SO-DNA adducts in vitro and in vivo by two different versions of (32)P-postlabelling/HPLC assays. When anionexchange cartridges were used for adduct enrichment the β-isomer of 7-substituted guanines was detected in in vitro SO-treated DNA as well as in mice lungs exposed to styrene at 750 and 1500 mg m(-3) for 21 days (6 h day(-1), 7 days week(-1)). In the lungs, the adduct levels were 6.5 and 23 per 10(8) nucleotides for the two doses, respectively. When the nuclease P1 resistant adducts were studied by the (32)P-postlabelling/HPLC assay involving nuclease P1/prostatic acid phosphatase hydrolysis, the main adducts in in vitro-treated DNA were the α-isomer of N(2)-substituted guanine, β-isomers of 1-substituted adenine and 3-substituted uracil. β1-SO-adenine adduct was detected in the mice lung tissues after conversion of the 1-substituted adduct to the βN(6)-SO-adenine adduct by the Dimroth rearrangement. The 1-adenine adduct levels for the two doses were found to be 0.17 and 0.51 per 10(8) nucleotides. The current results show the potential of using the 7-guanine and 1-adenine adducts as biomarkers in biomonitoring of styrene exposure.

The crystal and molecular structure of aminoguanidine monohydrochloride, CN4H7 +.CL- in which aminonoguanidine exists in the monocation form, was determined by single-crystal X-ray diffraction. The structure of the monocation is largely similar to that of aminoguanidine dication as present in previously studied divalent salts. The monocation was found to exist in the form of the tautom er that allows strong resonance in the guanyl group. As compared to the dication, the terminal hydrazine nitrogen atom bears one hydrogen atom less. The monocation is planar, the only atoms deviating from the plane being the hydrogens attached to the terminal hydrazine nitrogen. Quantum chemical calculations on the endiamine tautomer of aminoguanidine free base as well as on aminoguanidine monocation and dication were also perform ed by using the HF and MP2 ab initio methods and also the B3- LYP and B-LYP methods based on density functional theory. On the basis of the calculations, a predicted structure of the endiamine tautom er of aminoguanidine free base is presented

A reversed-phase (C18) HPLC method with diode-array detection was developed for the separation and determination of methylglyoxal bis(amidinohydrazone) (mitoguazone) and seven closely related aliphatic analogs thereof, namely the bis(amidinohydrazones) of glyoxal, dimethylglyoxal, ethylmethylglyoxal, methylpropylglyoxal, butylmethylglyoxal, diethylglyoxal and dipropylglyoxal. The mobile phase consisted of a non-linear binary gradient of methanol and 0.03 M aqueous sodium acetate buffer (pH 4.3). Good separation of the eight congeners was achieved. On increasing the methanol content of the eluent, the bis(amidinohydrazones) eluted in the order of increasing number of carbon atoms in the side-chains. The method was also applied to the quantitative analysis of the compounds in aqueous solution and, combined with ultrafiltration, for the separation of the eight congeners in spiked human blood serum. A separate simplified method for the quantitative determination of each of the compounds in spiked human blood serum samples was also developed. The methods developed made for the first time possible the simultaneous HPLC analysis of more than one bis(amidinohydrazones). The results obtained indicate that the bis(amidinohydrazones) studied obviously have a distinct tendency to form ion associates with acetate ions and probably also other carboxylate ions in aqueous solution. This aspect may be of biochemical significance, especially concerning the intracellular binding of the compounds. Each one of the compounds studied invariably gave rise to one peak only, this result supporting the theory that the conventional synthesis of each of the compounds gives rise to one geometrical isomer only. This result is completely in agreement with the results of previous proton and carbon NMR spectroscopic as well as X-ray diffraction studies.

The first crystallographic study on an aromatic analogue of the antileukemic agent methylglyoxal bis(amidinohydrazone) (MGBG) is reported. Thus, the crystal and molecular structures of two different geometrical isomers of phenylglyoxal bis(amidinohydrazone) (PhGBG) sulphate were determined by single-crystal X-ray diffraction. Crystals were prepared by recrystallizing PhGBG sulphate using either water or aqueous ethanol (volume ratio ethanol:water 1:4) as the solvent. Depending on the solvent, different types of crystals were obtained although the PhGBG sulphate employed was in the both cases from the same synthesis batch that had been prepared according to classical methods from the corresponding glyoxal. When a crystal obtained from water was studied, PhGBG was found to exist solely in the form of the anti-anti isomer. e. the same isomer that has been observed in the cases of all mono- and dialkylglyoxal bis(amidinohydrazones) so far studied. However, when PhGBG sulphate was recrystallized from 20 % aqueous ethanol, the crystals obtained consisted of a different geometrical isomer. In this anti-syn isomer the carbon-nitrogen double bond closest to the phenyl group had the syn configuration. In the anti-syn isomer, there is an internal hydrogen bond between the two amidinohydrazone moieties, which may markedly contribute to the stabilization of the isomer. The anti-syn isomer of PhGBG is analogous to the only isomer of trifluoromethylglyoxal bis(amidinohydrazone) so far observed. PhGBG sulphate constitutes the first case in which two different geometrical isomers of a bis(amidinohydrazone) have been observed. In the case of the much studied aliphatic mono- and dialkylglyoxal analogues, isomerization of the bis(amidinohydrazone) backbone has never been observed. The structural flexibility of the bis(amidinohydrazone) chain of PhGBG is obviously attributable to the electron-withdrawing resonance effect and perhaps also to the inductive and hyperconjugative effects of the phenyl group. The obviously facile isomerization of PhGBG may markedly influence the biochemical properties of the compound.

The first study on the infrared spectroscopy of the bis(amidinohydrazones) of various glyoxals is reported. The compounds studied include the antileukemic agents glyoxal bis(amidinohydrazone) and methylglyoxal bis(amidinohydrazone) (Mitoguazone) as well as seven mono-and dialkylglyoxal analogues thereof. Free bases as well as doubly protonated species (divalent salts) were investigated. Selectively deuterated analogues were also studied and were synthesized by exchanging nitrogen-bound hydrogen atoms for deuterium atoms. The effects of substituents, protonation and deuteration on the FT-IR spectra of the compounds are discussed.

Glyoxal bis(amidinohydrazone) (GBG) and several analogs thereof are compounds of considerable pharmacological interest, and a variety of HPLC and MECC methods have been developed for their analysis. In these methods, detection is invariably based on the strong UV absorption of the compound. Yet, almost nothing has been known of their UV and VIS spectral properties. In the present paper the UV and VIS Spectroscopy of GBG has been studied in several solvent systems (water, 0. 03 M aqueous sodium acetate buffer, 0. 1 inM aqueous NaOH and dimethylsulfoxide). In the case of solutions in bare water, the shape of the UV spectrum depends drastically on concentration, probably because of changes in the species distribution of GBG as a function of concentration. The spectrum comprises one maximum at ca. 200 nm, and between ca. 250 nin and 400 nm an absorption region with distinctly higher absorbance. In the case of aqueous sodium acetate as well as NaOH solutions, one strong maximum can be detected (at ca....

Aminoguanidine is not only an agent with a variety of pharmacological effects but also an important starting material of am idinohydrazone-type drugs and enzyme inhibitors. There­ fore, we have now synthesized am inoguanidine sulphate CN4H8 2+.SO4 2- and determined its structure by single-crystal X-ray diffraction. The doubly protonated (dication) form of amino­ guanidine that is present in the sulphate could, in principle, exist in the form of several different tautom ers. The crystal studied consisted exclusively of one tautomer: one of the nitrogens of the hydrazine moiety bears three hydrogen atoms while the other one (the one bound to the carbon) bears one hydrogen. The other two nitrogens are bound to two hydrogens each. The predominance of this tautomer can be explained by the very strong resonance in it. The dication of aminoguanidine is remarkably planar. The hydrogens of the hydrazine moeity are, however, clearly out of the plane of the other atoms. There is a strong hydrogen bond between the proton of the m onoprotonated nitrogen and one sulphate oxygen. This bond obviously causes the deviation of the hydrogen from the plane. The bonds between the carbon atom and the adjecnt nitrogens are essentially equally long, indicating that each bond has approximately the same amount of double bond character. One of the positive charges of the dication is thus delocalized, being shared by all of the atoms of the CN3 moiety. In this respect, the structure is similar to that of all bis(amidinohydrazones) whose structures have been determined. The other positive charge of aminoguanidine dication is localized at the nitrogen bearing three hydrogens.

Metabolic disposition of drugs and other xenobiotics includes glucuronidation reactions that are catalyzed by the uridine diphosphate glucuronosyltransferases (UGTs). The most common glucuronidation reactions are O- and N-glucuronidation and in this review, we discuss both, while the emphasis is on N-glucuronidation. Interspecies difference in glucuronidation is another central issue in this review due to its importance in drug development. Accordingly, the available data on glucuronidation in different animals comes mainly from the species that are used in preclinical studies to assess the safety of drugs under development. Both O- and N-glucuronidation reactions are chemically diverse. Different O-glucuronidation reactions are described and discussed, and many drugs that undergo such reactions are indicated. The compounds that undergo N-glucuronidation include primary aromatic amines, hydroxylamines, amides, tertiary aliphatic amines, and aromatic N-heterocycles. The interspecies variability in N-glucuronidation is particularly high, above all when it comes to aliphatic tertiary amines and aromatic N-heterocycles. The N-glucuronidation rates in humans are typically much higher than in animals, largely due to the activity of two enzymes, the extensively studied UGT1A4, and the more recently identified as a main player in N-glucuronidation, UGT2B10. We discuss both enzymes and review the findings that revealed the role of UGT2B10 in N-glucuronidation.

Compound 1 is an investigational, nanomolar inhibitor of catechol-O-methyltransferase (COMT) that suffers from poor oral bioavailability, most probably due to its low lipophilicity throughout most of the gastrointestinal tract and, to a lesser extent, its rapid systemic clearance. Several lipophilic esters were designed as prodrugs and synthesized in an attempt to optimize presystemic drug absorption. A modest twofold increase in 6-h exposure of 1 was observed with two prodrugs, compared to that of 1, after oral treatment in rats.

[formula: see text] Styrene 7,8-oxide (SO) induced N1-2'-deoxyadenosine 5'-phosphate (AMP) adducts deaminate to corresponding inosine derivatives. For the beta-isomer of N1-SO-AMP, the chiral alpha-carbon was found to be involved in the hydrolytic deamination, suggesting formation of an oxazolinium ring as an intermediate and that a water molecule attacks the benzylic carbon. The mechanism differs from the one suggested for the alpha-isomer of N1-SO-AMP, for which deamination occurs by direct attack of water at the 6-position of purine ring.

Medetomidine is a chiral imidazole derivate whose dextroenantiomer is pharmacologically active. The major metabolic pathway of dexmedetomidine [(+)-4-(S)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole] in humans is N-glucuronidation at the imidazolate nitrogens. We have purified the N3- and N1-glucuronides of dexmedetomidine, termed DG1 and DG2, respectively, according to their elution order in liquid chromatography and determined their structure by 1H nuclear magnetic resonance (NMR). Studying medetomidine glucuronidation by human liver microsomes (HLMs) and recombinant UDP glucuronosyltransferase (UGT) 1A4 indicated that another human UGT plays a major role in these activities. We now demonstrate that this enzyme is UGT2B10. HLMs catalyzed DG1 and DG2 formation, at a ratio of 3:1, with two-enzyme kinetics that contain both a high-affinity component, K(m1) values of 6.6 and 8.7 microM, and a low-affinity component, K(m2) values > 1 mM. The DG1/DG2 ratio in the case of UGT2B10 was lower, 1.4:1, whereas the substrate affinity for both reactions was high, K(m) values of 11 and 16 microM. UGT1A4 produced mainly DG1 (DG1/DG2 ratio of 6.6:1) at low substrate affinities, K(m) values above 0.6 mM, but superior expression-normalized V(max) values. Levomedetomidine [(-)-4-(R)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole] glucuronidation by HLMs yielded mostly the N3-glucuronide (LG1, structure determined by NMR), with monophasic kinetics and a K(m) value of 14 microM. The activity of UGT1A4 toward levomedetomide was low and generated both LG1 and LG2, whereas UGT2B10 exhibited relatively high activity and sharp regioselectivity, yielding only LG1, with a K(m) value of 7.4 microM. The results highlight the contribution of UGT2B10 to medetomidine glucuronidation and its potential importance for other N-glucuronidation reactions within the human liver.

OR-1896 is a pharmacologically active, long-lived metabolite of levosimendan. In the current study, the metabolism of (14)C-labelled OR-1896 was investigated in six healthy men after intravenous infusion over 10 min and in male rats after an intravenous bolus dose. In human plasma, the only (14)C-compounds detected were (14)C-OR-1896 and its deacetylated form, (14)C-OR-1855, in varying proportions in different subjects. In rat plasma >93% of radioactivity was associated with OR-1896. Radioactivity was mainly excreted to urine in both rats (about 69% of the dose) and humans (about 87% of the dose). OR-1896 was a major urinary compound in both humans and rats. Another major human metabolite was hypothesized as N-conjugated OR-1855. Other human and rat urinary biotransformation products were characterized as N-hydroxylated OR-1896 and N-hydroxylated OR-1855, as well as glucuronide or sulphate conjugates of N-hydroxyl OR-1896. The main difference between rat and human metabolism was a lower amount of OR-1855-related metabolites in the rats. In human faecal homogenates, only OR-1896 and OR-1855 were detected, whereas rat faecal metabolite profile was similar to that in urine.

The title compound, C7H18N82+.2Cl−.2H2O, has been found to exist as the anti-anti isomer, with an all-trans configuration of the bis(amidinohydrazone) chain, just like glyoxal bis(amidinohydrazone) and all its mono- and dialkylglyoxal analogues studied so far. The bis(amidinohydrazone) backbone of the dication is planar in contrast to the corresponding sulfate salt.

Styrene is an important industrial chemical that has shown genotoxicity in many toxicology assays. This is believed to be related to the DNA-binding properties of styrene-7,8-oxide (SO), a major metabolite of styrene. In this review, we have summarized knowledge on various aspects of styrene genotoxicity, especially in order to understand the formation and removal of primary DNA lesions, and the usefulness of biomarkers for risk assessment. Biological significances of specific DNA adducts and their role in the cascade of genotoxic events are discussed. Links between markers of external and internal exposure are evaluated, as well as metabolic aspects leading to the formation of DNA adducts and influencing biomarkers of biological effect. Finally, we suggest a design of a population study, which may contribute to our understanding genotoxic events in the exposure either to single xenobiotic or complex mixture.

Styrene is an extensively used industrial chemical that has been classified as a possible human carcinogen. The possible carcinogenicity may be related to the covalent DNA binding properties of styrene 7, 8-oxide, a major metabolite of styrene. We have developed a sensitive and a highly specific phosphorus-32-postlabeling method for the determination of 1-styrene 7, 8-oxide-adenine DNA adducts. These adducts were analyzed in white blood cells from workers exposed to styrene at mean level of 76.2 mg/m 3 . Three of nine exposed workers showed adducts above the detection limit; the mean was 0. 79 ± 0.14 1-styrene 7,8-oxide-adenine DNA adducts/10 9 nucleotides. None of the 11 control cells showed adducts above the detection limit, which was 0.4 adducts/10 9 nucleotides. The results show a potential of 1-adenine DNA adducts for predicting risks in the workers exposed to styrene.

The main achievements in the DNA adduct field in the 1990s have been technical innovations of methods for specific adducts reaching sensitivities required for low levels encountered in humans. Over 20 specific adducts or closely related groups of adducts have been determined in humans. The sources of the DNA-binding agents are endogenous and exogenous or both. In some of these studies adduct levels have been correlated to metabolic or DNA repair genotypes. An example of DNA adduct studies in human target tissue is taken on UV photoproducts in skin in situ. Adduct-induced mutations, specific mutation spectra, and their relationship to cancer are discussed. The quantitative adduct techniques will enable comparisons of endogenous and exogenous adduct levels and will give important clues to the etiology of human cancer. Furthermore, adducts will provide an intermediary tool for genotyping studies, both for metabolic enzyme and for DNA repair system genotypes. As the common polymorphisms are likely to cause at most moderate increases in the risk of cancer, the intermediary adduct endpoint is a necessary proof of causal relationships. The present and future biomonitoring studies will cover many endpoints to link the mechanistic steps from DNA adducts to cancer via mutations and modulating host susceptibility factors.

Alkyl epoxides are important intermediates in the chemical industry. They are also formed in vivo during the detoxification of alkenes. Alkyl epoxides have shown genotoxicity in many toxicology assays which has been associated with their covalent binding to DNA. Here aspects of the formation and properties of DNA adducts, induced by some industrially important alkenes and mono-substituted epoxides are discussed. These include propylene oxide, epichlorohydrin, allyl glycidyl ether and the epoxy metabolites of styrene and butadiene. The major DNA adducts formed by epoxides are 7-substituted guanines, 1- and 3-substituted adenines and 3-substituted cytosines. In addition, styrene oxide and butadiene monoepoxide are able to modify exocyclic sites in the DNA bases, the sites being in the case of styrene oxide N(2)- and O(6)-positions of guanine, N(6)-adenine as well as N(4)-and O(2)-cytosine. In vivo the main adduct is the 7-substituted guanines. The 1-substituted adenines have also shown marked levels, and these adducts should also be targets in biomonitoring of human exposures. Due to its low mutagenicity, 7-substituted guanines are considered as a surrogate marker for other mutagenic lesions, e.g. those of 1-adenine or 3-uracil adducts.

Propylene oxide, a widely used monofunctional alkylating agent, has been shown to be genotoxic in in vitro test systems and induces tumors in the nasal tissues of experimental animals. Propylene oxide, like related alkylating agents, forms several different adducts with DNA bases, but predominantly at the 7-position of guanine. We have previously described the in vitro and in vivo formation and stability of this major adduct. The aim of the present study was to perform a similar investigation of other adducts of propylene oxide. 1-(2-Hydroxypropyl)adenine (1-HP-adenine) and 3-(2-hydroxypropyl)cytosine (3-HP-cytosine), as well as their rearrangement products to N(6)-(2-hydroxypropyl)adenine (N(6)-HP-adenine) and 3-(2-hydroxypropyl)uracil (3-HP-uracil), respectively, were analysed by a very sensitive (32)P-postlabelling method involving nuclease P1 enhancement and radioisotope detector-coupled HPLC separation. All four adducts could be detected in DNA treated in vitro with propylene oxide. The sum of the levels of 1- and N(6)-HP-adenine amounted to 3.5% and the sum of 3-HP-cytosine and 3-HP-uracil to 1.7%, respectively, of 7-(2-hydroxypropyl)guanine (7-HP-guanine). In male Fischer 344 rats exposed to 500 p.p.m. propylene oxide by inhalation for 20 days, 1-HP-adenine was detected in all analysed tissues, including nasal epithelium, lung and lymphocytes, whereas N(6)-HP-adenine was only found in the tissues of the nasal cavities. The highest level of 1-HP-adenine (2.0 mol/10(6) mol of normal nucleotides, i.e. 2% of 7-HP-guanine) was found in the respiratory nasal epithelium, which also represents the major target for tumour induction in the rat following inhalation of propylene oxide. The levels of this adduct in the lung and in the lymphocytes were considerably lower, amounting to 15 and 9%, respectively, of that of the respiratory nasal epithelium. In rats killed 3 days after cessation of exposure, practically no decrease in 1-HP-adenine was observed, indicating no or very slow repair. 3-HP-uracil could only be detected in the respiratory nasal epithelia of propylene-exposed rats and its concentration was as low as 0.02 mol/10(6) mol of normal nucleotides (0.02% of 7-HP-guanine). Since 3-HP-uracil was chemically much more stable than the latter, the obtained animal data suggest repair of the cytosine and/or uracil adducts. Incubation of propylene oxide-reacted DNA with a protein extract from mammalian cells indicated that an enzymatic repair mechanism exists for removal of 3-HP-cytosine, but not for 3-HP-uracil or 1- and N(6)-HP-adenine. Another finding was that uracil glycosylase is probably not involved. The level of 1-HP-adenine in the propylene oxide-exposed rats was approximately 50 times lower than that of 7-HP-guanine. Nevertheless, this adduct is conveniently analysed and has high chemical stability and recovery, which results in high sensitivity (detection limit 0.3 mol/10(9) mol of normal nucleotides using 10 microgram DNA). 1-HP-adenine might, therefore, be considered as an alternative to 7-HP-guanine for monitoring exposure to propylene oxide.

DNA binding of tamoxifen and some structurally-related drugs (toremifene, clomiphene, triparanol and raloxifene) in rat liver was studied using the 32 P-postlabelling method. As only tamoxifen was shown to form high levels of DNA adducts, the identity of these adducts was studied. Recently, we have identified by mass spectroscopy the two main tamoxifen adducts in rat liver DNA as the N -desmethyltamoxifen and tamoxifen adducts of N 2 -deoxyguanosine in which the linkage is through α-carbon in the tamoxifen structure. Minor adducts were identical to different diastereomers of α-( N 2 -deoxyguanosinyl)tamoxifen and of α-( N 6 -deoxyadenosinyl)tamoxifen. Altogether these adducts accounted for at least 95% of adducts formed in vivo, implicating that the α-hydroxylation of the ethyl group is the major activation pathway for DNA adducts.

Epoxybutanediol is one of the epoxide metabolites of butadiene (BD). A pair of diastereomeric N-1-adenine adducts were formed by reacting epoxybutanediol with deoxyadenosine 5′-monophosphate (5′-dAMP). These two N-1-adenine adducts rearranged in a base-catalysed reaction to an N6-trihydroxybutyl-adenine adduct, which was characterized by UV and mass spectroscopy. Using the 32P-postlabelling/HPLC assay the same adducts were detected in diepoxybutane (DEB)-treated DNA in vitro and in liver DNA samples from rats exposed to BD by inhalation. Adenine adducts of epoxybutanediol are probably suitable for monitoring BD exposure.

Tamoxifen-induced DNA adducts were searched in leucocyte DNA from breast cancer patients. Total white blood cell DNA from tamoxifen-treated and control patients was analysed by 32 P-postlabelling using HPLC-radioactivity detection. Rat liver DNA was used as a positive standard. In blinded analysis four of the six treated patients showed DNA adducts; none of the five controls were positive. The identity of fraction as a tamoxifen adduct was confirmed by using different chromatographic systems, each with spiked rat liver samples. The level of adducts in the treated patients was 5.5 adducts/10 9 nucleotides as compared to an apparent level of 1.9/10 9 in the controls.