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.

Mental Health of Healthcare Workers during COVID-19 Pandemic in Taiwan: The First Wave Outbreak Occurred 1 Year Later Than in Other Countries.

We probed the psychological influence exerted on traumatic stress endured by healthcare workers (HCWs) and the coping behaviors adopted during the first wave of COVID-19 in Taiwan, which occurred one year later than in other countries. Clinical HCWs from two branches of a hospital network in Taichung, Taiwan, were recruited for this cross-sectional study. The participants were administered a questionnaire on sociodemographic and work-related characteristics, perceived influence exerted by COVID-19, coping behaviors in relation to COVID-19, and Impact of Event Scale-Revised scores. We obtained 769 valid questionnaires. A chi-square test, generalized linear modeling, and multivariate stepwise regression analyses were performed. Although the first wave of COVID-19 occurred one year later in Taiwan than in other countries, the traumatic stress experienced by Taiwanese HCWs was noted to be comparable to that of those in other countries. Factors for increased traumatic stress included caring for more patients with COVID-19, fair or poor self-rated mental health, higher perceived influence of COVID-19, vulnerable household income, and more negative coping behaviors. Positive coping behaviors such as exposure reduction and protection measures decreased traumatic stress. Accordingly, managers should strengthen protective measures, enhance COVID-19-related training, and provide psychological support and counseling for high-risk employees.

Does direct settlement of intra-province medical reimbursements improve financial protection among middle-aged and elderly population in China? Evidence based on CHARLS data.

In low- and middle-income countries, social health insurance schemes are the main focus of efforts to achieve universal health coverage (UHC) by promoting access to health care and financial protection. Problems with financial protection in China are caused mainly by health insurance fragmentation and a rapid rise in medical expenditure. In this context, China implemented a policy of direct settlement of intra-provincial medical reimbursement in 2014. We evaluated the impact of the policy on financial protection with a population aged 45 and above based on the China Health and Retirement Longitudinal Study from 2011 to 2018. We estimated the policy effects using the difference-in-differences method, based on coarsened exact matching. We found that the policy significantly reduced the catastrophic health expenditures (CHEs) rate by approximately 10% in the population, whether middle-aged or elderly. Subgroup analyses indicated that middle-aged and elderly people living in western China and with lower household incomes received greater protection from the policy. The CHEs rate for the two age groups in western China was reduced by 16.26% and 20.12%, respectively. The CHEs rate was reduced by 24.51% and 17.32% for middle-aged individuals in the lowest and second household income quartiles, respectively, and by 21.31% for older adults in the second household income quartile. The new rural cooperative medical scheme exerted a smaller protective effect than urban medical insurance among the participants aged 60 and older. We found that in addition to optimizing health insurance schemes, more health care reform measures, such as adopting more efficient payment methods and rationalizing medical expenditures, should be combined to help reduce health inequities and accelerate progress toward achieving UHC and the Sustainable Development Goals.

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.

UV and temperature effects on chloroacetanilide and triazine herbicides degradation and cytotoxicity.

The purpose of this study was to explore the stability and toxicity of the herbicides and their degradation byproduct after exposure to different environmental factors. Triazines (atrazine, propazine, simazine) and chloroacetanilides (acetochlor, alachlor, metolachlor) which are commonly used herbicides were evaluated for cytotoxicity in different UV (254 nm and 365 nm) and temperature (4 °C, 23 °C, and 40 °C) conditions as well as degradation rates. Atrazine with the highest LD50 (4.23 µg mL-1) was less toxic than the other tested triazine herbicides Chloroacetanilides tested were more toxic than tested triazines, with LD50 0.08-1.42 µg mL-1 vs 1.44-4.23 µg mL-1, respectively. Alachlor with LD50 0.08 µg mL-1 showed the strongest toxic response as compared with other tested herbicides. Temperatures only did not alter cytotoxicity of the tested herbicides, except for acetochlor and alachlor showing about 45 % more cell death after exposure to 40 °C for 2 h. At all 3 tested temperatures, 2 h of UV treatments did not affect cytotoxic effects of the tested herbicides, except for acetochlor and alachlor. At 4 °C, acetochlor toxicity was attenuated about 63 % after UV 365 nm exposure; but alachlor toxicity was enhanced after either UV 254 or 365 nm exposure for about 40 % and 24 %, respectively. At 23 °C, acetochlor toxicity was enhanced about 35 % after UV 254 nm exposure, but attenuated about 48 % after UV 365 nm exposure. Alachlor toxicity was enhanced about 34 % after UV 254 nm and 23 °C exposure. In combination of UV 254 nm and 40 °C, acetochlor toxicity was lowered by 63 % and alachlor toxicity was no change as compared with 4 °C, no UV group. After co-treatment with UV 365 nm and 40 °C both acetochlor and alachlor toxicity was enhanced 55 % and 80 %, respectively. Through degradation analysis by LC-MS/MS, alachlor showed the most dramatic degradation (only 0.58 %-10.58 % remaining) after heat and UV treatments.

Use of personal protective equipment against coronavirus disease 2019 by healthcare professionals in Wuhan, China: cross sectional study.

OBJECTIVE: To examine the protective effects of appropriate personal protective equipment for frontline healthcare professionals who provided care for patients with coronavirus disease 2019 (covid-19). DESIGN: Cross sectional study. SETTING: Four hospitals in Wuhan, China. PARTICIPANTS: 420 healthcare professionals (116 doctors and 304 nurses) who were deployed to Wuhan by two affiliated hospitals of Sun Yat-sen University and Nanfang Hospital of Southern Medical University for 6-8 weeks from 24 January to 7 April 2020. These study participants were provided with appropriate personal protective equipment to deliver healthcare to patients admitted to hospital with covid-19 and were involved in aerosol generating procedures. 77 healthcare professionals with no exposure history to covid-19 and 80 patients who had recovered from covid-19 were recruited to verify the accuracy of antibody testing. MAIN OUTCOME MEASURES: Covid-19 related symptoms (fever, cough, and dyspnoea) and evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, defined as a positive test for virus specific nucleic acids in nasopharyngeal swabs, or a positive test for IgM or IgG antibodies in the serum samples. RESULTS: The average age of study participants was 35.8 years and 68.1% (286/420) were women. These study participants worked 4-6 hour shifts for an average of 5.4 days a week; they worked an average of 16.2 hours each week in intensive care units. All 420 study participants had direct contact with patients with covid-19 and performed at least one aerosol generating procedure. During the deployment period in Wuhan, none of the study participants reported covid-19 related symptoms. When the participants returned home, they all tested negative for SARS-CoV-2 specific nucleic acids and IgM or IgG antibodies (95% confidence interval 0.0 to 0.7%). CONCLUSION: Before a safe and effective vaccine becomes available, healthcare professionals remain susceptible to covid-19. Despite being at high risk of exposure, study participants were appropriately protected and did not contract infection or develop protective immunity against SARS-CoV-2. Healthcare systems must give priority to the procurement and distribution of personal protective equipment, and provide adequate training to healthcare professionals in its use.

Preparedness of medical education in China: Lessons from the COVID-19 outbreak.

The COVID-19 outbreak can be seen as a 'big test' for China; a summative assessment of its preparedness on multiple fronts, including medical education. Being intimately involved in the coordinated response, the First Affiliated Hospital of Sun Yat-sen University has been a first-hand witness to the strengths and weaknesses of the current medical education system in China. On the one hand, we believe that the distinguished contributions in disease containment efforts by healthcare professionals indicated that our medical education system has achieved its intended outcomes and is socially accountable. On the other hand, we have also identified three major issues that need to be addressed from an educational standpoint: insufficient emphasis on public health emergency preparedness; unsophisticated mechanisms for interdisciplinary cooperation; and inadequate guidance in medical ethics. Whilst these reflections might be seen in its summative form, we would suggest changing it to that of a formative process, where we learn from our assessment through observation and feedback of the gaps, upon which improvement of our present situation can be made. We hope that these lessons may be helpful to our colleagues in the rest of China and around the world, who are engaged in medical educational reform.

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.

Wastewater analysis for nicotine, cocaine, amphetamines, opioids and cannabis in New York City.

According to current surveys and overdoses data, there is a drug crisis in the USA. Wastewater-based epidemiology (WBE) is an evolving discipline that analyses wastewater samples to detect drugs and metabolites to estimate drug consumption in a certain community. This study demonstrates how drug relative presence could be tracked by testing wastewater, providing real-time results, in different boroughs in New York City throughout 1 year. We developed and fully validated two analytical methods, one for 21 drugs and metabolites, including nicotine, cocaine, amphetamines, opioids and cannabis markers; and another for the normalization factor creatinine. Both methods were performed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using positive electrospray ionization, achieving a limit of quantification of 5-10 ng/L for drugs and metabolites, and 0.01 mg/L for creatinine. These methods were applied to 48 one-time grab wastewater samples collected from six wastewater treatment plants in New York City (Manhattan, The Bronx, Queens and Brooklyn), eight different times throughout 2016, before and after major holidays, including Memorial Day, 4th of July, Labour Day and New Year's. In this study, the drug group normalized concentrations present in the wastewater samples, in decreasing order, were cocaine, nicotine, opioids, cannabis and amphetamines. When looking at individual compounds, the one with the highest normalized concentration was benzoylecgonine (BE), followed by cotinine, morphine and 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH). To estimate community use, these concentrations were multiplied by the corresponding correction factor, and the most present were THCCOOH, followed by BE, cotinine and morphine. When comparing the treatment plants by drug group (nicotine, cocaine, amphetamines, opioids and cannabis), samples collected from The Bronx had the highest normalized concentrations for nicotine, cocaine and opioids; The Bronx and Manhattan for cannabis; and Manhattan and Queens for amphetamines. In most of the cases, no effect due to holiday was observed. This study provides the first snapshot of drug use in New York City and how that changes between key calendar dates employing wastewater analysis.

Copper(II)-Catalyzed Selective Hydroboration of Ketones and Aldehydes.

A novel nonanuclear copper(II) complex obtained by a facile one-pot self-assembly was found to catalyze the hydroboration of ketones and aldehydes with the absence of an activator under mild, solvent-free conditions. The catalyst is air- and moisture-stable, displaying high efficiency (1980 h-1 turnover frequency, TOF) and chemoselectivity on aldehydes over ketones and ketones over imines. This represents a rare example of divalent copper catalyst for the hydroboration of carbonyls.

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.

ANTENNA, a Multi-Rank, Multi-Layered Recommender System for Inferring Reliable Drug-Gene-Disease Associations: Repurposing Diazoxide as a Targeted Anti-Cancer Therapy.

Existing drug discovery processes follow a reductionist model of "one-drug-one-gene-one-disease," which is inadequate to tackle complex diseases involving multiple malfunctioned genes. The availability of big omics data offers opportunities to transform drug discovery process into a new paradigm of systems pharmacology that focuses on designing drugs to target molecular interaction networks instead of a single gene. Here, we develop a reliable multi-rank, multi-layered recommender system, ANTENNA, to mine large-scale chemical genomics and disease association data for prediction of novel drug-gene-disease associations. ANTENNA integrates a novel tri-factorization based dual-regularized weighted and imputed One Class Collaborative Filtering (OCCF) algorithm, tREMAP, with a statistical framework based on Random Walk with Restart and assess the reliability of specific predictions. In the benchmark, tREMAP clearly outperforms the single-rank OCCF. We apply ANTENNA to a real-world problem: repurposing old drugs for new clinical indications without effective treatments. We discover that FDA-approved drug diazoxide can inhibit multiple kinase genes responsible for many diseases including cancer and kill triple negative breast cancer (TNBC) cells efficiently [Formula: see text]. TNBC is a deadly disease without effective targeted therapies. Our finding demonstrates the power of big data analytics in drug discovery and developing a targeted therapy for TNBC.

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.

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.

Plant Translation Initiation Complex eIFiso4F Directs Pokeweed Antiviral Protein to Selectively Depurinate Uncapped Tobacco Etch Virus RNA.

Pokeweed antiviral protein (PAP) is a ribosome inactivating protein (RIP) that depurinates the sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis. PAP depurinates viral RNA, and in doing so, lowers the infectivity of many plant viruses. The mechanism by which PAP accesses uncapped viral RNA is not known, impeding scientists from developing effective antiviral agents for the prevention of the diseases caused by uncapped RNA viruses. Kinetic rates of PAP interacting with tobacco etch virus (TEV) RNA, in the presence and absence of eIFiso4F, were examined, addressing how the eIF affects selective PAP targeting and depurination of the uncapped viral RNA. PAP-eIFs copurification assay and fluorescence resonance energy transfer demonstrate that PAP forms a ternary complex with the eIFiso4G and eIFiso4E, directing the depurination of uncapped viral RNA. eIFiso4F selectively targets PAP to depurinate TEV RNA by increasing PAP's specificity constant for uncapped viral RNA 12-fold, when compared to the depurination of an oligonucleotide RNA that mimics the SRL of large rRNA, and cellular capped luciferase mRNA. This explains how PAP is able to lower infectivity of pokeweed viruses, while preserving its own ribosomes and cellular RNA from depurination: PAP utilizes cellular eIFiso4F in a novel strategy to target uncapped viral RNA. It may be possible to modulate and utilize these PAP-eIFs interactions for their public health benefit; by repurposing them to selectively target PAP to depurinate uncapped viral RNA, many plant and animal diseases caused by these viruses could be alleviated.

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.

Quantitative analysis of opioids and cannabinoids in wastewater samples.

Wastewater-based epidemiology is an innovative approach that uses the analysis of human excretion products in wastewater to obtain information about exposure to drugs in defined population groups. We developed and validated an analytical method for the simultaneous determination of opioids (morphine, oxycodone, hydrocodone, oxymorphone and hydromorphone), and cannabinoids (Δ9-tetrahydrocannabinol, 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) and THCCOOH-glucuronide) in raw-influent wastewater samples by ultra-high performance liquid chromatography-tandem mass spectrometry. Method validation included linearity (5-1 000 ng/L for opioids, 10-1 000 ng/L for cannabinoids), imprecision (<21.2%), accuracy (83%-131%), matrix effect (from -35.1% to -14.7%) and extraction efficiency (25%-84%), limit of detection (1-5 ng/L) and quantification (5-10 ng/L) and auto-sampler stability (no loss detected). River and wastewater samples were collected in triplicate from different locations in New York City and stored at -20 °C until analysis. Water from sewage overflow location tested positive for morphine (10.7 ng/L), oxycodone (4.2-23.5 ng/L), oxymorphone (4.8 ng/L) and hydromorphone (4.2 ng/L). Raw influent wastewater samples tested positive for morphine (133.0-258.3 ng/L), oxycodone (31.1-63.6 ng/L), oxymorphone (16.0-56.8 ng/L), hydromorphone (6.8-18.0 ng/L), hydrocodone (4.0-12.8 ng/L) and THCCOOH (168.2-772.0 ng/L). This method is sensitive and specific for opioids and marijuana determination in wastewater samples.

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.

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.