Insight Into Factors Governing Formation, Synthesis and Stereochemical Configuration of DNA Adducts Formed by Mitomycins.

Mitomycin C, (MC), an antitumor drug used in the clinics, is a DNA alkylating agent. Inert in its native form, MC is reduced to reactive mitosenes in cellulo which undergo nucleophilic attack by DNA bases to form monoadducts as well as interstrand crosslinks (ICLs). These properties constitute the molecular basis for the cytotoxic effects of the drug. The mechanism of DNA alkylation by mitomycins has been studied for the past 30 years and, until recently, the consensus was that drugs of the mitomycins family mainly target CpG sequences in DNA. However, that paradigm was recently challenged. Here, we relate the latest research on both MC and dicarbamoylmitomycin C (DMC), a synthetic derivative of MC which has been used to investigate the regioselectivity of mitomycins DNA alkylation as well as the relationship between mitomycins reductive activation pathways and DNA adducts stereochemical configuration. We also review the different synthetic routes to access mitomycins nucleoside adducts and oligonucleotides containing MC/DMC DNA adducts located at a single position. Finally, we briefly describe the DNA structural modifications induced by MC and DMC adducts and how site specifically modified oligonucleotides have been used to elucidate the role each adduct plays in the drugs cytotoxicity.

Cytotoxicity, crosslinking and biological activity of three mitomycins.

While interstrand crosslinks (ICLs) have been considered as one type of DNA damage in the past, there is mounting evidence suggesting that these highly cytotoxic lesions are processed differently by the cellular machinery depending upon the ICL structure. In this study, we examined the crosslinking ability of three mitomycins, the structure of the ICLs they produce and the cytotoxicity of the drugs toward three different cell lines. The drugs are: mitomycin C (1), decarbamoylmitomycin C (2), and a mitomycin-conjugate (3) whose mitosane moiety is linked to a N-methylpyrrole carboxamide. We found that, overall, both MC and compound 3 show strong similarities regarding their alkylation of DNA, while DMC alkylating behavior is markedly different. To gain further insight into the mode of action of these drugs, we performed high throughput gene expression and gene ontology analysis to identify gene expression and cellular pathways most impacted by each drug treatment in MCF-7 cell lines. We observed that the novel mitomycin derivative (3) specifically causes changes in the expression of genes encoding proteins involved in cell integrity and tissue structure. Further analysis using bioinformatics (IPA) indicated that the new derivative (3) displays a stronger downregulation of major signaling networks that regulate the cell cycle, DNA damage response and cell proliferation when compared to MC and DMC. Collectively, these findings demonstrate that cytotoxic mechanisms of all three drugs are complex and are not solely related to their crosslinking abilities or the structure of the ICLs they produce.

Synthesis of Oligonucleotides Containing Trans Mitomycin C DNA Adducts at N6 of Adenine and N2 of Guanine.

Mitomycin C, (MC), an antitumor drug, is a DNA alkylating agent currently used in the clinics. Inert in its native form, MC is reduced to reactive mitosenes, which undergo nucleophilic attack by guanine or adenine bases in DNA to form monoadducts as well as interstrand crosslinks (ICLs). Although ICLs are considered the most cytotoxic lesions, the role of each individual adduct in the drug's cytotoxicity is still not fully understood. Synthetic routes have been developed to access modified oligonucleotides containing dG MC-monoadducts and dG-MC-dG ICL at a single position of their base sequences to investigate the biological effects of these adducts. However, until now, oligonucleotides containing monoadducts formed by MC at the adenine base had not been available, thus preventing the examination of the role played by these lesions in the toxicity of MC. Here, we present a route to access these substrates. Structural proof of the adducted oligonucleotides were provided by enzymatic digestion to nucleosides and high-resolution mass spectral analysis. Additionally, parent oligonucleotides containing a dG monoadduct and a dG-MC-dG ICL were also produced. The stability and physical properties of all substrates were compared via CD spectroscopy and UV melting temperature studies. Finally, virtual models were created to explore the conformational space and structural features of these MC-DNA complexes.

Synthesis of Oligonucleotides containing the cis-Interstrand Crosslink Produced by Mitomycins in their Reaction with DNA.

Mitomycin C (MC) an antitumor drug and decarbamoylmitomycin C (DMC), a derivative of MC lacking the carbamoyl moiety, are DNA alkylating agents which can form DNA interstrand crosslinks (ICLs) between deoxyguanosine residues located on opposing DNA strands. MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α, trans) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-ß, cis). The crosslinking reaction is diastereospecific: trans-crosslinks are formed exclusively at CpG sequences, while cis-crosslinks are formed only at GpC sequences. Until now, oligonucleotides containing 1"-ß-deoxyguanosine adducts or ICL at a specific site could not be synthesized, thus limiting the investigation of the role played by the stereochemical configuration at C1'' in the toxicity of these compounds. Here, a novel biomimetic synthesis to access these substrates is presented. Structural proof of the adducted oligonucleotides and ICL were provided by enzymatic digestion to nucleosides, high resolution mass spectral analysis, CD spectroscopy and UV melting temperature studies. Finally, a virtual model of the 25-mer 1"-ß ICL synthesized was created to explore the conformational space and structural features of the crosslinked duplex.

Synthesis of Mitomycin C and decarbamoylmitomycin C N6 deoxyadenosine-adducts.

Mitomycin C (MC), an anti-cancer drug, and its analog, decarbamoylmitomycin C (DMC), are DNA-alkylating agents. MC is currently used in the clinics and its cytotoxicity is mainly due to its ability to form Interstrand Crosslinks (ICLs) which impede DNA replication and, thereby, block cancer cells proliferation. However, both MC and DMC are also able to generate monoadducts with DNA. In particular, we recently discovered that DMC, like MC, can form deoxyadenosine (dA) monoadducts with DNA. The biological role played by these monoadducts is worthy of investigation. To probe the role of these adducts and to detect them in enzymatic digests of DNA extracted from culture cells treated by both drugs, we need access to reference compounds i.e. MC and DMC dA-mononucleoside adducts. Previous biomimetic methods used to generate MC and DMC mononucleoside adducts are cumbersome and very low yielding. Here, we describe the diastereospecific chemical synthesis of both C-1 epimers of MC and DMC deoxyadenosine adducts. The key step of the synthesis involves an aromatic substitution reaction between a 6-fluoropurine 2'-deoxyribonucleoside and appropriately protected stereoisomeric triaminomitosenes to form protected-MC-dA adducts with either an S or R stereochemical configuration at the adenine-mitosene linkage. Fluoride-based deprotection methods generated the final four reference compounds: the two stereoisomeric MC-dA adducts and the two stereoisomeric DMC-dA adducts. The MC and DMC-dA adducts synthesized here will serve as standards for the detection and identification of such adducts formed in the DNA of culture cells treated with both drugs.

Interdependent Sequence Selectivity and Diastereoselectivity in the Alkylation of DNA by Decarbamoylmitomycin†C.

Mitomycin C (MC), an antitumor drug, and decarbamoylmitomycin C (DMC), a derivative of MC, alkylate DNA and form deoxyguanosine monoadducts and interstrand crosslinks (ICLs). Interestingly, in mammalian culture cells, MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-ß). The molecular basis for the stereochemical configuration exhibited by DMC has been investigated using biomimetic synthesis. Here, we present the results of our studies on the monoalkylation of DNA by DMC. We show that the formation of 1"-ß-deoxyguanosine adducts requires bifunctional reductive activation of DMC, and that monofunctional activation only produces 1"-α-adducts. The stereochemistry of the deoxyguanosine adducts formed is also dependent on the regioselectivity of DNA alkylation and on the overall DNA CG content. Additionally, we found that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by mitomycins: At 0 °C, both deoxyadenosine (dA) and deoxyguanosine (dG) alkylation occur whereas at 37 °C, mitomycins alkylate dG preferentially. The new reaction protocols developed in our laboratory to investigate DMC-DNA alkylation raise the possibility that oligonucleotides containing DMC 1"-ß-deoxyguanosine adducts at a specific site may be synthesized by a biomimetic approach.

Sequence-Dependent Diastereospecific and Diastereodivergent Crosslinking of DNA by Decarbamoylmitomycin C.

Mitomycin C (MC), a potent antitumor drug, and decarbamoylmitomycin C (DMC), a derivative lacking the carbamoyl group, form highly cytotoxic DNA interstrand crosslinks. The major interstrand crosslink formed by DMC is the C1'' epimer of the major crosslink formed by MC. The molecular basis for the stereochemical configuration exhibited by DMC was investigated using biomimetic synthesis. The formation of DNA-DNA crosslinks by DMC is diastereospecific and diastereodivergent: Only the 1''S-diastereomer of the initially formed monoadduct can form crosslinks at GpC sequences, and only the 1''R-diastereomer of the monoadduct can form crosslinks at CpG sequences. We also show that CpG and GpC sequences react with divergent diastereoselectivity in the first alkylation step: 1"S stereochemistry is favored at GpC sequences and 1''R stereochemistry is favored at CpG sequences. Therefore, the first alkylation step results, at each sequence, in the selective formation of the diastereomer able to generate an interstrand DNA-DNA crosslink after the "second arm" alkylation. Examination of the known DNA adduct pattern obtained after treatment of cancer cell cultures with DMC indicates that the GpC sequence is the major target for the formation of DNA-DNA crosslinks in vivo by this drug.

Isolation and Rationale for the Formation of Isomeric Decarbamoylmitomycin C- N6-deoxyadenosine Adducts in DNA.

Mitomycin C (MC) is an anticancer agent that alkylates DNA to form monoadducts and interstrand cross-links. Decarbamoylmitomycin C (DMC) is an analogue of MC lacking the carbamate on C10. The major DNA adducts isolated from treatment of culture cells with MC and DMC are N2-deoxyguanosine (dG) adducts and adopt an opposite stereochemical configuration at the dG-mitosene bond. To elucidate the molecular mechanisms of DMC-DNA alkylation, we have reacted short oligonucleotides, calf thymus, and M. luteus DNA with DMC using biomimetic conditions. These experiments revealed that DMC is able to form two stereoisomeric deoxyadenosine (dA) adducts with DNA under bifuntional reduction conditions and at low temperature. The dA-DMC adducts formed were detected and quantified by HPLC analysis after enzymatic digestion of the alkylated DNA substrates. Results revealed the following rules for DMC dA alkylation: (i) DMC dA adducts are formed at a 48- to 4-fold lower frequency than dG adducts, (ii) the 5'-phosphodiester linkage of the dA adducts is resistant to snake venom diesterase, (iii) end-chain dA residues are more reactive than internal ones in duplex DNA, and (iv) nucleophilic addition by dA occurs on both faces of DMC and the ratio of stereoisomeric dA adducts formed is dependent on the end bases located at the 3' or 5' position. A key finding was to discover that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by DMC: at 0 °C, both dA and dG alkylation occur, whereas at 37 °C, DMC preferentially alkylates dG residues.

Acetone promoted 1,4-migration of an alkoxycarbonyl group on a syn-1,2-diamine.

A 2-protected cis-amino mitosene undergoes an irreversible acetone promoted isomerization and converts to the 1-isomer. Kinetic studies and DFT calculations of the reaction are reported. An organocatalytic mechanism is proposed, involving a covalent intermediate formed by reaction of the mitosene and acetone.

Comparative Error-Free and Error-Prone Translesion Synthesis of N(2)-2'-Deoxyguanosine Adducts Formed by Mitomycin C and Its Metabolite, 2,7-Diaminomitosene, in Human Cells.

Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates DNA upon reductive activation. 2,7-Diaminomitosene (2,7-DAM) is a major metabolite of MC in tumor cells, which also alkylates DNA. MC forms seven DNA adducts, including monoadducts and inter- and intrastrand cross-links, whereas 2,7-DAM forms two monoadducts. Herein, the biological effects of the dG-N(2) adducts formed by MC and 2,7-DAM have been compared by constructing single-stranded plasmids containing these adducts and replicating them in human embryonic kidney 293T cells. Translesion synthesis (TLS) efficiencies of dG-N(2)-MC and dG-N(2)-2,7-DAM were 38 ± 3 and 27 ± 3%, respectively, compared to that of a control plasmid. This indicates that both adducts block DNA synthesis and that dG-N(2)-2,7-DAM is a stronger replication block than dG-N(2)-MC. TLS of each adducted construct was reduced upon siRNA knockdown of pol η, pol κ, or pol ζ. For both adducts, the most significant reduction occurred with knockdown of pol κ, which suggests that pol κ plays a major role in TLS of these dG-N(2) adducts. Analysis of the progeny showed that both adducts were mutagenic, and the mutation frequencies (MF) of dG-N(2)-MC and dG-N(2)-2,7-DAM were 18 ± 3 and 10 ± 1%, respectively. For both adducts, the major type of mutation was G → T transversions. Knockdown of pol η and pol ζ reduced the MF of dG-N(2)-MC and dG-N(2)-2,7-DAM, whereas knockdown of pol κ increased the MF of these adducts. This suggests that pol κ predominantly carries out error-free TLS, whereas pol η and pol ζ are involved in error-prone TLS. The largest reduction in MF by 78 and 80%, respectively, for dG-N(2)-MC and dG-N(2)-2,7-DAM constructs occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down. This result strongly suggests that, unlike pol κ, these three TLS polymerases cooperatively perform the error-prone TLS of these adducts.

Synthesis of Mitomycin C and Decarbamoylmitomycin C N(2) deoxyguanosine-adducts.

Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) - a derivative of MC lacking the carbamate on C10 - are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine-mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment.

Mitomycin C and its analog trigger cytotoxicity in MCF-7 and K562 cancer cells through the regulation of RAS and MAPK/ERK pathways.

Mitomycin C (MC) is an anti-cancer drug which functions by forming interstrand crosslinks (ICLs) between opposing DNA strands. MC analog, 10-decarbamoyl mitomycin C (DMC), unlike MC, has stronger cytotoxic effects on cancer cells with TP53 mutation. We previously demonstrated that MC/DMC could activate p21WAF1/CIP1 in MCF-7 (TP53-proficient) and K562 (TP53 deficient) cells in a TP53-independent mode. We also found that MC/DMC regulate AKT activation in a TP53-dependent manner and that AKT deactivation is not associated with the activation of p21WAF1/CIP1 in response to MC/DMC treatment. RAS proteins are known players in the upstream mediated signaling of p21WAF1/CIP1 activation that leads to control of cell proliferation and cell death. Thus, this prompted us to investigate the effect of both drugs on the expression of RAS proteins and regulation of the MAPK/ERK signaling pathways in MCF-7 and K562 cancer cells. To accomplish this goal, we performed comparative label free proteomics profiling coupled to bioinformatics/complementary phosphoprotein arrays and Western blot validations of key signaling molecules. The MAPK/ERK pathway exhibited an overall downregulation upon MC/DMC treatment in MCF-7 cells but only DMC exhibited a mild downregulation of that same pathway in TP53 mutant K562 cells. Furthermore, treatment of MCF-7 and K562 cell lines with oligonucleotides containing the interstrand crosslinks (ICLs) formed by MC or DMC shows that both ICLs had a stronger effect on the downregulation of RAS protein expression in mutant TP53 K562 cells. We discuss the implication of this regulation of the MAPK/ERK pathway in relation to cellular TP53 status.

Synthesis of a major mitomycin C DNA adduct via a triaminomitosene.

We report here the synthesis of two amino precursors for the production of mitomycin C and 10-decarbamoylmitomycin C DNA adducts with opposite stereochemistry at C-1. The triamino mitosene precursors were synthesized in 5 steps from mitomycin C. In addition synthesis of the major mitomycin C-DNA adduct has been accomplished via coupling of a triaminomitosene with 2-fluoro-O(6)-(2-p-nitrophenylethyl)deoxyinosine followed by deprotection at the N(2) and O(6) positions.

Repair of mitomycin C mono- and interstrand cross-linked DNA adducts by UvrABC: a new model.

Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending. The mechanisms and consequences of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. Using DNA fragments containing a MC-mono-dG or an ICL dG-MC-dG adduct, we found (i) UvrABC incises only at the strand containing MC-mono-dG adducts; (ii) UvrABC makes three types of incisions on an ICL dG-MC-dG adduct: type 1, a single 5' incision on 1 strand and a 3' incision on the other; type 2, dual incisions on 1 strand and a single incision on the other; and type 3, dual incisions on both strands; and (iii) the cutting kinetics of type 3 is significantly faster than type 1 and type 2, and all of 3 types of cutting result in producing DSB. We found that UvrA, UvrA+UvrB and UvrA+UvrB+UvrC bind to MC-modified DNA specifically, and we did not detect any UvrB- and UvrB+UvrC-DNA complexes. Our findings challenge the current UvrABC incision model. We propose that DSBs resulted from NER of ICL dG-MC-dG adducts contribute to MC antitumor activity and mutations.

Synthesis of an oligodeoxyribonucleotide adduct of mitomycin C by the postoligomerization method via a triamino mitosene.

The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA monofunctionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known structures. The synthesis of one of the monoadducts (8) by the postoligomerization method was accomplished both on the nucleoside and oligonucleotide levels, the latter resulting in the site-specific placement of 8 in a 12-mer oligodeoxyribonucleotide 26. This is the first application of this method to the synthesis of a DNA adduct of a complex natural product. Preparation of the requisite selectively protected triaminomitosenes 14 and 24 commenced with removal of the 10-carbamoyl group from MC, followed by reductive conversion to 10-decarbamoyl-2,7-diaminomitosene 10. This substance was transformed to 14 or 24 in several steps. Both were successfully coupled to the 2-fluoro-O(6)-(2-trimethylsilylethyl)deoxyinosine residue of the 12-mer oligonucleotide. The N(2)-phenylacetyl protecting group of 14 after its coupling to the 12-mer oligonucleotide could not be removed by penicillinamidase as expected. Nevertheless, the Teoc protecting group of 24 after coupling to the 12-mer oligonucleotide was removed by treatment with ZnBr2 to give the adducted oligonucleotide 26. However, phenylacetyl group removal was successful on the nucleoside-level synthesis of adduct 8. Proof of the structure of the synthetic nucleoside adduct included HPLC coelution and identical spectral properties with a natural sample, and (1)H NMR. Structure proof of the adducted oligonucleotide 26 was provided by enzymatic digestion to nucleosides and authentic adduct 8, as well as MS and MS/MS analysis.

Involvement of Akt in mitomycin C and its analog triggered cytotoxicity in MCF-7 and K562 cancer cells.

Mitomycin C (MC) is a well-known DNA alkylating agent. MC analog, 10-decarbamoyl mitomycin C (DMC), unlike MC, has stronger effects on cancer with p53 mutation. We previously demonstrated that MC/DMC could activate p21WAF1/CIP1 in MCF-7 (p53-proficient) and K562 (p53-deficient) cells in a p53-independent mode. This study aimed to elucidate the upstream signaling pathway of p21WAF1/CIP1 activation triggered by MC/DMC. Besides p53, Akt plays an important role on deactivating p21WAF1/CIP1 . The results showed that MC/DMC inhibited Akt in MCF-7 cells, but not in K562 cells. By knocking down p53, the Akt inhibition in MCF-7 cells was alleviated. This implied that the deactivated Akt caused by MC/DMC was p53-dependent. With Akt activator (SC79), p21WAF1/CIP1 activation triggered by MC/DMC in MCF-7 cells was not reduced. This indicated that Akt inhibition triggered by MC/DMC was not associated with MC/DMC-induced p21WAF1/CIP1 activation. Label-free quantitative proteomic profiling analysis revealed that DMC has a stronger effect on down-regulating the PI3K/Akt signaling pathway in MCF-7 cells as compared to MC. No significant effect of MC/DMC on PI3K/Akt in K562 cells was observed. In summary, MC/DMC regulate Akt activation in a p53-dependent manner. This Akt deactivation is not associated with p21WAF1/CIP1 activation in response to MC/DMC.

Mitomycin C and decarbamoyl mitomycin C induce p53-independent p21WAF1/CIP1 activation.

Mitomycin C (MC), a commonly used anticancer drug, induces DNA damage via DNA alkylation. Decarbamoyl mitomycin C (DMC), another mitomycin lacking the carbamate at C10, generates similar lesions as MC. Interstrand cross-links (ICLs) are believed to be the lesions primarily responsible for the cytotoxicity of MC and DMC. The major ICL generated by MC (α-ICL) has a trans stereochemistry at the guanine-drug linkage whereas the major ICL from DMC (ß-ICL) has the opposite, cis, stereochemistry. In addition, DMC can provoke strong p53-independent cell death. Our hypothesis is that the stereochemistry of the major unique ß-ICL generated by DMC is responsible for this p53-independent cell death signaling. p53 gene is inactively mutated in more than half of human cancers. p21WAF1/CIP1 known as a major effector of p53 is involved in p53-dependent and -independent control of cell proliferation and death. This study revealed the role of p21WAF1/CIP1 on MC and DMC triggered cell damage. MCF-7 (p53-proficient) and K562 (p53-deficient) cells were used. Cell cycle distributions were shifted to the G1/S phase in MCF-7 treated with MC and DMC, but were shifted to the S phase in K562. p21WAF1/CIP1 activation was observed in both cells treated with MC and DMC, and DMC triggered more significant activation. Knocking down p53 in MCF-7 did not attenuate MC and DMC induced p21WAF1/CIP1 activation. The α-ICL itself was enough to cause p21WAF1/CIP1 activation.

Plasma concentrations of ropivacaine following ultrasound-guided or nerve-stimulator-guided femoral nerve block: A prospective randomised study.

OBJECTIVE: Our aim was to establish a plasma concentration curve for ropivacaine following femoral nerve blockade and to ascertain whether the resulting plasma concentrations differ significantly depending on whether neurostimulation (NS) or ultrasound (US) guidance was used. METHODS: Sixteen male and female subjects aged 18 to 80 who were scheduled to undergo unilateral total knee replacement or anterior cruciate ligament reconstruction under general or spinal anaesthesia, and for whom a femoral nerve block was indicated for postoperative analgesia, were enrolled in this prospective, randomised study. Patients were randomised to undergo either US or NS-guidance femoral nerve blocks. All blocks were performed with 20 mL of 5mg/mL ropivacaine. Blood samples were drawn before the nerve block and 20, 30, 40, 50, 60, 70, and 80 minutes after the block. Plasma levels of ropivacaine were analysed by high performance liquid chromatography (HPLC). RESULTS: All blocks were successful and no patient showed signs or symptoms of local anaesthetic toxicity. The plasma concentration of ropivacaine peaked at 30 minutes in both arms. There was no significant difference in peak levels between US and NS-guidance (0.325±0.186 versus 0.356±0.106 µg/mL). Cmax and tmax were very similar between groups (0.364±0.177 versus 0.344±0.127 µg/mL, 33.75±15.06 versus 31.25±13.56 min for US and NS, respectively). CONCLUSION: Plasma concentrations of ropivacaine peak around 30 minutes after a femoral nerve block regardless of the technique used. No significant difference was found between US- and NS-guidance.

Report on the analysis of common beverages spiked with gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL) using NMR and the PURGE solvent-suppression technique.

In forensic evidence, the identification and quantitation of gamma-hydroxybutyric acid (GHB) in "spiked" beverages is challenging. In this report, we present the analysis of common alcoholic beverages found in clubs and bars spiked with gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL). Our analysis of the spiked beverages consisted of using (1)H NMR with a water suppression method called Presaturation Utilizing Relaxation Gradients and Echoes (PURGE). The following beverages were analyzed: water, 10% ethanol in water, vodka-cranberry juice, rum and coke, gin and tonic, whisky and diet coke, white wine, red wine, and beer. The PURGE method allowed for the direct identification and quantitation of both compounds in all beverages except red and white wine where small interferences prevented accurate quantitation. The NMR method presented in this paper utilizes PURGE water suppression. Thanks to the use of a capillary internal standard, the method is fast, non-destructive, sensitive and requires no sample preparation which could disrupt the equilibrium between GHB and GBL.

Differential toxicity of DNA adducts of mitomycin C.

The clinically used antitumor agent mitomycin C (MC) alkylates DNA upon reductive activation, forming six covalent DNA adducts in this process. This paper focuses on differential biological effects of individual adducts in various mammalian cell cultures, observed in the authors' laboratories. Evidence is reviewed that various adducts are capable of inducing different cell death pathways in cancer cells. This evidence is derived from a parallel study of MC and its derivatives 2,7-diaminomitosene (2,7-DAM) which is the main metabolite of MC and forms two monoadducts with DNA, and decarbamoyl mitomycin C (DMC), which alkylates and crosslinks DNA, predominantly with a chirality opposite to that of the DNA adducts of MC. Specifically, 2,7-DAM is not cytotoxic and does not activate the p53 pathway while MC and DMC are cytotoxic and able to activate the p53 pathway. DMC is more cytotoxic than MC and can also kill p53-deficient cells by inducing degradation of Checkpoint 1 protein, which is not seen with MC treatment of the p53-deficient cells. This difference in the cell death pathways activated by the MC and DMC is attributed to differential signaling by the DNA adducts of DMC. We hypothesize that the different chirality of the adduct-to-DNA linkage has a modulating influence on the choice of pathway. Future studies will be directed to elucidate mechanisms of MC- and DMC-DNA adduct signaling in a structure-dependent context.