Metabolism of oxazaphosphorines

The cancer therapy using cyclophosphamide (CP) has been associated with adverse effects on the testicular function that raises concerns about the future fertility potential among cancer survivors. Curcumin, a polyphenol, has shown to possess a plethora of biological functions including tissue protective effects. In the present study, we investigated the protective effects of curcumin nanocrystals (NC) in mitigation of CP-induced testicular toxicity. Healthy adult (8–10 week) and prepubertal (2 week) male Swiss albino mice were injected with a single dose of CP (200 mg/kg) intraperitoneally (i.p). NC (4 mg/kg, i.p.) was administered every alternate day, for 35 days in adult mice while, a single dose of NC was injected intraperitoneally to prepubertal mice 1 h prior to CP. Administration of multiple doses of NC ameliorated CP-induced testicular toxicity in adult mice, which was evident from the improved sperm functional competence, sperm chromatin condensation, seminiferous tubule architecture and decreased apoptosis in testicular cells. Further, administration of NC 1 h prior to CP in prepubertal mice modulated the expression of genes pertaining to proliferation, pluripotency, DNA damage and DNA repair in spermatogonial cells at 24 h after the treatment. Overall, these results suggest that NC could be a promising chemoprotective agent, which can have potential application in male fertility preservation.

The DNA alkylating prodrug cyclophosphamide (CPA), alone or in combination with other agents, is one of the most commonly used anti-cancer agents. As a prodrug, CPA is activated by cytochrome P450 2B6 (CYP2B6), which is transcriptionally regulated by the human constitutive androstane receptor (hCAR). Therefore, hCAR agonists represent novel sensitizers for CPA-based therapies. Among known hCAR agonists, compound 6-(4-chlorophenyl)imidazo-[2,1-b]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is the most potent and broadly utilized in biological studies. Through structural modification of CITCO, we have developed a novel compound DL5016 (32), which has an EC₅₀ value of 0.66 μM and E_MAX value of 4.9 when activating hCAR. DL5016 robustly induced the expression of hCAR target gene CYP2B6, at both the mRNA and protein levels, and caused translocation of hCAR from the cytoplasm to the nucleus in human primary hepatocytes. The effects of DL5016 were highlighted by dramatically enhancing the efficacy of CPA-based cytotoxicity to non-Hodgkin lymphoma cells.

The increased number of cell divisions undergone by spermatogonia of older fathers cannot fully account for the observed increase in germline genetic damage. Studies have shown that the mechanisms induced in germ cells in response to oxidative damage varies with age, that DNA repair efficiency declines, and both sperm DNA damage and spontaneous mutations increase. However, it is not known whether the altered response with age is a cause, or consequence, of an age-associated change in cell susceptibility to genetic damage.

Following a single 150 mg/kg dose of cyclophosphamide (CP), young (8-weeks old) and aged (17-month old) male mice were examined 24 h later for induced genetic damage in epididymal spermatozoa using the alkaline comet and sperm chromatin stability assays. Apoptosis among testicular cells was examined on tissue cross-sections using the TUNEL assay.

Sperm showed no significant increase in DNA strand breaks with age (detected by the comet assay) and no change in sperm chromatin stability (detected by the SCSA assay). Following CP treatment, there was no effect on DNA-strand breakage but sperm chromatin instability was significantly higher. Furthermore, it was also significantly elevated in old treated, compared with young treated, animals suggesting that increased age affects the sensitivity of epididymal sperm to chromatin damage.

There was no difference in apoptosis in testicular germ cells from either young or old control animals, while CP administration resulted in a significant increase in apoptosis among young animals but not old animals. Following genotoxin exposure, an increase in chromatin instability in the spermatozoa of old animals and a decrease in the ability of their testicular germ cells undergo apoptosis suggests an age-related decrease in genome protection mechanisms. Since those germ cells are only transiently present in the testis, it is likely that this age-related deterioration originates in the spermatogonial stem cells. The findings are also evidence that the safety evaluation of reproductive genotoxins should consider young and old individuals separately.

Preclinical drug safety assessment includes in vitro studies with physiologically relevant cell cultures. As an in vitro system for hepatic toxicology testing, we have been generating cell clones of human hepatoblastoma cell line HepG2 by lentiviral transduction of phase I cytochrome P450 (CYP) enzymes. Here, we present a stable CYP2C19-overexpressing HepG2 cell clone (HepG2-2C19 C1) showing an enzyme activity of approximately 82 pmol x min⁻¹ x mg⁻¹ total cellular protein. The phenotypic stability over several passages of HepG2-2C19 C1 renders them to be a suitable reference cell clone for benchmarking CYP2C19 enzyme activity. In addition, we were interested to analyze acute cytotoxicity of the model drug cyclophosphamide (CPA) metabolized by HepG2-2C19 C1 and by a previously generated CYP3A4-overexpressing HepG2 cell clone. Upon 10 mM CPA exposure, we were able to detect its metabolites 4-hydroxy-cyclophosphamide and acrolein in CYP3A4- and CYP2C19-expressing cell clones, but not in parental HepG2 cell line. XTT and ATP assays showed a modest reduction of cell viability of not more than 50% with high dose (10 mM) CPA treatment. By contrast, dramatic acute cytotoxic effects of CPA were evident by the formation of nuclear γH2AX foci and by increased cell death events. These effects were paralleled by substantial decreases of cell membrane integrity as measured by the trypan blue exclusion test. Our data on CYP enzyme overexpressing HepG2 cell clones clearly show that cytotoxicity of CPA is dramatically underestimated by standard metabolic activity tests. Thus, additional tests to quantitate DNA damage formation and cell death induction might be required to realistically assess cytotoxicity of such compounds.

‘Nitro­gen mustard’ bis­(2-chloro­eth­yl)amine derivatives (2R,4S,5R)- and (2S,4S,5R)-2-[bis­(2-chloro­eth­yl)amino]-3,4-dimethyl-5-phenyl-1,3,2-oxaza­phos­pho­lidin-2-one (2a and 2b, respectively), C₁₄H₂₁Cl₂N₂O₂P, and (2R,4R)- and (2S,4R)-2-[bis­(2-chloro­eth­yl)amino]-4-isobutyl-1,3,2-oxaza­phospho­lidin-2-one (3a and 3b, respectively), C₁₀H₂₁Cl₂N₂O₂P, were synthesized as a mixture of diastereomers through a 1:1 reaction of enanti­omerically pure chiral amino alcohols with bis­(2-chloro­eth­yl)phospho­ramidic dichloride. Flash column chromatography yielded diastereomerically pure products, as supported by ³¹P NMR. The crystal structures of 2b and 3b were obtained to determine their absolute configuration at phospho­rus, and ³¹P NMR chemical shift trends are proposed based on the spatial relationship of the bis­(2-chloro­eth­yl)amine moiety and the chiral substituent of the amino alcohol. Oxaza­phospho­lidinones were observed to have a more downfield ³¹P NMR chemical shift when the aforementioned substituents are in a syn configuration and vice versa for when they are anti.

Halogenated hydrocarbons (HHCs) encompass a broad group of aliphatic and aromatic chemicals. Many are environmental contaminants or industrial solvents. This article focuses on select HHCs that are known to exhibit nephrotoxicity in animals and/or humans. Chemicals that are reviewed include select examples from haloalkanes, haloacids, haloalkenes, halogenated aromatics, halogenated ethers (which include some commonly used anesthetic agents), and ifosfamide and cyclophosphamide. The article is divided into processes involved in nephrotoxicity. The first major section summarizes metabolic pathways for bioactivation and/or detoxification. Metabolism comprises two major pathways: cytochrome P450 (CYP)-dependent oxidation, which can result in bioactivation or generate substrates for Phase II conjugation reactions; and glutathione-dependent bioactivation pathways, which involve subsequent metabolism by cysteine conjugate beta-lyase or flavin-containing monooxygenase. The next major section summarizes key features of acute and chronic nephrotoxicity, which typically involve single dose or short-term, high-dose exposures for acute and long-term, low-dose exposures for chronic. The final section summarizes select human health risk assessments for HHCs.