Search

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is an organophosphate ester-based flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) reliably disrupts cytosine methylation from cleavage (2 hpf) through early-gastrulation (6 hpf). Therefore, the objective of this study was to determine whether TDCIPP-induced effects on cytosine methylation persist beyond 6 hpf. First, we exposed embryos to vehicle or TDCIPP from 0.75 hpf to 6, 24, or 48 hpf, and then conducted bisulfite amplicon sequencing of a target locus (lmo7b) using genomic DNA derived from whole embryos. Within both vehicle- and TDCIPP-treated embryos, CpG methylation was similar at 6 hpf and CHG/CHH methylation were similar at 24 and 48 hpf (relative to 6 hpf). However, relative to 6 hpf within the same treatment, CpG methylation was lower within vehicle-treated embryos at 48 hpf and TDCIPP-treated embryos at 24 and 48 hpf - an effect that was driven by acceleration of CpG hypomethylation. Similar to our previous findings with DNA methyltransferase, we found that, even at high μM concentrations, TDCIPP had no effect on zebrafish and human thymine DNA glycosylase activity (a key enzyme that decreases CpG methylation), suggesting that TDCIPP-induced effects on CpG methylation are not driven by direct interaction with thymine DNA glycosylase. Finally, using 5-methylcytosine (5-mC)-specific whole-mount immunochemistry and automated imaging, we found that exposure to TDCIPP increased 5-mC abundance within the yolk of blastula-stage embryos, suggesting that TDCIPP may impact cytosine methylation of maternally loaded mRNAs during the maternal-to-zygotic transition. Overall, our findings suggest that TDCIPP disrupts the trajectory of cytosine methylation during zebrafish embryogenesis, effects which do not appear to be driven by direct interaction of TDCIPP with key enzymes that regulate cytosine methylation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cidofovir (CDF) and its cyclic analogue (cCDF) have shown potential in vitro and in vivo antiviral activity against cytomegalovirus (CMV) retinitis. However, hydrophilic nature of CDF may affect cell permeation across lipophilic epithelium and thus limit its effectiveness in the treatment of CMV retinitis. In the present study, we have tested a novel hypothesis, which involves chemical derivatization of cCDF into lipophilic transporter-targeted prodrug [via conjugation with different carbon chain length of lipid raft and targeting moiety (biotin) for sodium-dependent multivitamin transporter (SMVT)]. We have synthesized and characterized three derivatives of cCDF including biotin B-C2-cCDF, B-C6-cCDF, and B-C12-cCDF. Physicochemical properties such as solubility, partition coefficient (n-octanol/water and ocular tissue), bioreversion kinetics, and interaction with SMVT transporter have been determined. Among these novel conjugates, B-C12-cCDF has shown higher interaction to SMVT transporter with lowest half maximal inhibitory concentration value, higher cellular accumulation, and high tissue partitioning. Improvement in physicochemical properties, lipophilicity, and interaction with transporter was observed in the trend of increasing the lipid chain length, that is, B-C12-cCDF > B-C6-cCDF > B-C2-cCDF. These results indicate that transporter-targeted lipid analogue of cCDF exhibits improved cellular accumulation along with higher transporter affinity and hence could be a viable strategy for the treatment of CMV retinitis., (Copyright © 2012 Wiley Periodicals, Inc.)

The synthesis of the unsaturated 4,6-dideoxy-3-fluoro-2-keto-beta-d-glucopyranosyl nucleosides of 5-fluorouracil (6a), N(6)-benzoyl adenine (6b), uracil (6c), thymine (6d) and N(4)-benzoyl cytosine (6e), is described. Monoiodination of compounds 1a,b, followed by acetylation, catalytic hydrogenation and finally regioselective 2'-O-deacylation afforded the partially acetylated dideoxynucleoside analogues of 5-fluorouracil (5a) and N(6)-benzoyl adenine (5b), respectively. Direct oxidation of the free hydroxyl group at the 2'-position of 5a,b, with simultaneous elimination reaction of the beta-acetoxyl group, afforded the desired unsaturated 4,6-dideoxy-3-fluoro-2-keto-beta-D-glucopyranosyl derivatives 6a,b. Compounds 1c-e were used as starting materials for the synthesis of the dideoxy unsaturated carbonyl nucleosides of uracil (6c), thymine (6d) and N(4)-benzoyl cytosine (6e). Similarly a protection-selective deprotection sequence followed by oxidation of the free hydroxyl group at the 2'-position of the dideoxy benzoylated analogues 9c-e with simultaneous elimination reaction of the beta-benzoyl group, gave the desired nucleosides 6c-e. None of the compounds was inhibitory to a broad spectrum of DNA and RNA viruses at subtoxic concentrations. The 5-fluorouracil derivative 6a was more cytostatic (50% inhibitory concentration ranging between 0.2 and 12 microM) than the other compounds., (Copyright 2009 Elsevier Inc. All rights reserved.)

Esterification of cidofovir (CDV), an antiviral nucleoside phosphonate, with alkyl or alkoxyalkyl groups increases antiviral activity by enhancing cell uptake and conversion to CDV diphosphate. Hexadecyloxypropyl-CDV (HDP-CDV) has been shown to be 40-100 times more active than CDV in vitro in cells infected with herpes group viruses, variola, cowpox, vaccinia or ectromelia viruses. Since the first phosphorylation of CDV may be rate limiting, we synthesized the hexadecyloxypropyl-phosphate (HDP-P-) and octadecyloxyethyl-phosphate (ODE-P-) conjugates of CDV and phosphonomethoxy-ethyl-adenine (PMEA, adefovir). We tested the CDV analogs in cells infected with human cytomegalovirus, herpes simplex virus, cowpox virus and vaccinia virus; the analogs of PMEA were tested in cells infected with the human immunodeficiency virus, type 1. In general, the alkoxyalkyl-phosphate conjugates of CDV were substantially more active than CDV. HDP-P-CDV and ODE-P-CDV were 4.6-40 times more active against HCMV and 7-30 times more active against cowpox and vaccinia in vitro. Although the compounds of this type were more cytotoxic than the unmodified bases, their selectivity for virally infected cells was generally greater than the parent nucleotides except that HDP-P-PMEA showed little or no selectivity in HIV-1 infected MT-2 cells. Although the new compounds with an interposed phosphate were generally less active than the corresponding alkoxyalkyl esters of CDV and PMEA, the present approach provides a possible alternative method for enhancing the antiviral activity of drugs of this class.

Acyclic nucleoside phosphonates (ANPs) and in particular (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, cidofovir, CDV, Vistide) and its adenine counterpart (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA] are highly active against orf virus infections. This parapoxvirus commonly causes infection in sheep, goats, but also humans. Alkoxyalkyl esters of CDV have an increased oral bioavailability and are more active against orthopoxviruses than the parent compounds. In the present study, the potency of several alkoxyalkyl esters of CDV, cyclic cidofovir (cCDV) and (S)-HPMPA was evaluated against different orf virus isolates in two cell types, human embryonic lung (HEL) fibroblast and primary lamb keratinocytes. Each prodrug was at least 10-fold more active than its parent compound in both cell types. Of all the compounds tested, the (S)-HPMPA alkoxyalkyl esters showed the highest activity and selectivity against orf virus. Our results support the development of alkoxyalkyl esters of ANPs as antivirals not only for the treatment of complicated human orf lesions, but also in the therapy and prophylaxis of contagious ecthyma in sheep and goats.

A retrospective study was performed to collect information regarding efficacy and toxicity of cidofovir (CDV) in allogeneic stem cell transplant patients. Data were available on 82 patients. The indications for therapy were cytomegalovirus (CMV) disease in 20 patients, primary preemptive therapy in 24 patients, and secondary preemptive therapy in 38 patients. Of the patients, 47 had received previous antiviral therapy with ganciclovir, foscarnet, or both drugs. The dosage of CDV was 1 to 5 mg/kg per week followed by maintenance every other week in some patients. The duration of therapy ranged from 1 to 134 days (median, 22 days). All patients received probenecid and prehydration. Ten of 20 (50%) patients who were treated for CMV disease (9 of 16 with pneumonia) responded to CDV therapy, as did 25 of 38 (66%) patients who had failed or relapsed after previous preemptive therapy and 15 of 24 (62%) patients in whom CDV was used as the primary preemptive therapy. Of the patients, 21 (25.6%) developed renal toxicity that remained after cessation of therapy in 12 patients. Fifteen patients developed other toxicities that were potentially due to CDV or the concomitantly given probenecid. No toxicity was seen in 45 (61.6%) patients. Cidofovir can be considered as second-line therapy in patients with CMV disease failing previous antiviral therapy. However, additional studies are needed before CDV can be recommended for preemptive therapy.

The induction of SCE by ribo- and deoxyribonucleosides of 8-hydroxyguanine, 5-hydroxycytosine, and 2-hydroxyadenine was tested using human peripheral blood lymphocytes. All of these compounds caused an increase in SCE frequency. The potency of SCE induction was as follows: 5-OH-C, 5-OH-dC > 8.OH-G > 8-OH-dG > 2-OH-A, 2-OH-dA. These results suggest that the oxidized nucleosides are incorporated into DNA with different efficiencies (or are repaired with different efficiencies) and exhibit genotoxicity in human blood cells. Ribo- and deoxyribo-derivatives of 5-OH-Cyt and 2-OH-Ade also showed mutagenic activity in Salmonella typhimurium TA 100.

Ethenocytosine (epsilon C) is a highly mutagenic exocyclic DNA lesion induced by carcinogens vinyl chloride and urethane. We have examined base incorporation and extension at a site-specific epsilon C residue by a quantitative gel electrophoretic assay using an exonuclease-deficient version of Escherichia coli DNA polymerase I (Klenow fragment) as the model enzyme. The data show that the KM for incorporation of adenine or thymine opposite epsilon C by is about 5 orders of magnitude higher than that for the incorporation of guanine opposite normal cytosine. The KM for base extension past epsilon C:A and epsilon C:T pairs is 1-2 orders of magnitude higher than that observed for a C:G pair. Although adenine misinsertion is favored over that of thymine, base extension occurs more readily when the base incorporated opposite epsilon C is thymine.

The nucleoside analogue (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) inhibited the replication of herpes simplex virus (HSV) types 1 and 2 in tissue culture cells at about 1.0 micrograms/ml, whereas Acyclovir (ACV) had an EC50 of about 0.10-0.50 micrograms/ml. The purpose of these studies was to evaluate the efficacy of topically applied HPMPC in animal models of primary and recurrent genital HSV-2 infections. Mice treated with 5%, 1% or 0.5% HPMPC three times daily, beginning 6 or 24 h after virus inoculation had reduced vaginal viral replication regardless of time of initiation of therapy. ACV at 5% also reduced vaginal viral replication, but not as effectively as HPMPC. In primary infection of guinea pigs, therapy with 5% or 1% HPMPC beginning at 24 h but not 72 h significantly altered lesion development. However, 5% HPMPC was highly toxic to guinea pigs. Vaginal viral replication was reduced significantly with either 1% or 0.3% HPMPC initiated at 24 h. In these studies, HPMPC was also more efficacious than 5% ACV. Topical treatment with 1% HPMPC did not reduce the incidence or severity of spontaneous or UV-induced recurrent genital lesions. These results indicate that topical therapy with 1%, 0.5% or 0.3% HPMPC was more effective than 5% ACV in the treatment of primary genital HSV-2 infections of guinea pigs and mice and suggest that HPMPC should be considered for topical use in humans.

A reproducible pattern of respiratory disease was produced in calves inoculated intranasally with a pathogenic strain of bovine herpesvirus-1 (BHV-1). A latent infection was established which could be reactivated by means of corticosteroid administration. Groups of calves were given a single dose of 20 mg/kg of (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine (HPMPC) either the day before or the day following virus inoculation. The drug markedly reduced clinical signs and virus replication; the therapeutic dose appeared to be more effective than the dose given one day before virus inoculation. The establishment of latency was not prevented and a single dose of HPMPC, the day before a course of dexamethasone (6 weeks after the acute infection), did not prevent virus shedding.

Bropirimine is a biological response modifier (BRM) with potential antineoplastic and antiviral indications. Recent results have documented the negative findings in the Ames Salmonella assay, the in vitro UDS assay and the mouse lymphoma TK+/- assay as well as positive findings in the in vitro cytogenetic assay in CHO cells. Extensive mechanistic studies failed to establish the reason for positive findings in the in vitro cytogenetic assays. The data reported here cast doubt on the relevance of the in vitro cytogenetic results and suggest limited in vivo genotoxic potential. At doses as high as 150 mg/kg (i.p.) and 6.73 g/kg (p.o.), no evidence of chromosome aberration induction was observed in rat bone marrow cytogenetic assays. Consistent with these data, plasma and bone marrow tissue levels in similarly treated animals were well below those required for activity in the in vitro chromosome aberration assays. Positive results were obtained in the mouse micronucleus assay. However, the significance of these findings may be explained by markedly different pathways of metabolism in that species as compared to the rat. Hence, the findings in the mouse are of questionable relevance to human risk assessment. Exposure of humans to bropirimine, under therapeutically acceptable regimens is unlikely to constitute a genotoxic health hazard.

Bropirimine (U-54,461) is a novel compound which is being developed as a biological response modifier for use in treatment of neoplastic and viral disease. Compounds of this type exert their therapeutic effects by immuno-stimulation or other non-cytotoxic mechanisms. The purpose of the experiments described in this paper was to evaluate the hazard potential of this drug. Bropirimine was previously reported to be negative in the Ames Salmonella assay (Aaron et al., 1989a) and the in vitro UDS assay (Aaron et al., 1989b). In experiments reported here positive response was observed in a test for clastogenicity in vitro in CHO cells, but bropirimine was negative in the L5178Y mouse lymphoma TK+/- assay. A subsequent experiment demonstrated the ability of bropirimine to induce HPRT mutations in CHO cells. Interestingly, evidence for induction of chromosome aberrations in the L5178Y cells by bropirimine was also obtained. While the reason for the apparent insensitivity of the L5178Y TK+/- assay to bropirimine is unexplained by the experiments, it is clear that at high dose bropirimine is capable of clastogenesis in both CHO and L5178Y cells and can give rise to gene mutations in CHO cells but apparently not in L5178Y cells.

Bropirimine is an immunomodulator and has been investigated as an antineoplastic drug as well as for antiviral indications. However, the standard of prudence and the level of concern necessary in safety assessment in the alternative therapeutic situations, namely, antineoplastic therapy as opposed to treatment of non-life-threatening viral illnesses, is dramatically different. In previous reports from this laboratory, bropirimine was shown to be non-mutagenic in the Ames Salmonella assay (Aaron et al., 1989b), the in vitro UDS assay (Aaron et al., 1989a) and the L5178Y TK+/- assay but positive in the CHO cell chromosome aberration assay, in the presence of S9 (Aaron et al., 1991a). In this manuscript, we provide data gathered in attempts to further characterize the apparent requirement for S9 and understand the mechanism by which bropirimine induces chromosome aberrations. For example, heat inactivation of the S9 significantly reduced, but did not eliminate, the aberration induction. In addition, collection of mitotic cells without use of colcemid failed to reduce the aberration yield. Furthermore, no evidence of S9-mediated activation of bropirimine to an electrophilic, macromolecular binding species was observed in vitro, nor did lysosomal toxicity appear to contribute to the effect. Several analogs were tested for clastogenic potential; the 5-chloro analog was also clastogenic, but not the 5-iodo-, 5-bromo-3-fluorophenyl- or non-halogenated analogs. Thus, the mechanism of aberration induction remains obscure, but we have confirmed the need for presence of exogenous protein in order for the clastogenicity of bropirimine to be manifest and have ruled out several non-threshold mechanisms for toxicity.