Metabolite Of Cyclophosphamide Research Articles

Zebrafish teratogenicity test with metabolic activation (m DarT): Effects of phase I activation of acetaminophen on zebrafish Danio rerio embryos

The zebrafish Danio rerio embryo test with metabolic activation (m DarT) was developed to assess the teratogenic effects of proteratogens. In this study induced rat liver microsomes (RLM) were used as a mammalian metabolic activation system (MAS), since they contain various cytochrome P450 (CYP) isoforms at high concentrations. Acetaminophen (APAP) is considered not to be teratogenic in vivo, however, in vitro teratogenic effects were observed, e.g. in rat whole embryo culture. The CYP2E1 activation of APAP to the reactive metabolite N-acetyl- p-benzoquinone imine (NAPQI) mainly occurs, when the glucuronidation and sulfatation pathways are saturated. In vivo the soft electrophile NAPQI is usually inactivated by hepatic reduced glutathione (GSH), a soft nucleophile. In this study, we investigated the teratogenic and lethal effects of APAP after CYP activation in zebrafish embryos. In the test groups with APAP and metabolic activation 11.7 ± 7.6% (2 mM), 25.0 ± 8.7% (4 mM) and 50.0 ± 21.8% (6 mM) affected embryos were seen, reaching statistical significance at 4 mM APAP. When embryos were exposed to 6 mM APAP, MAS and 3 mM GSH the percentage of affected embryos decreased to 6.7 ± 5.8%. In contrast teratogenic and lethal effects of metabolically activated cyclophosphamide (CPA) could not be prevented by GSH addition, because the CPA metabolites are strong electrophiles, which preferentially bind to hard nucleophiles like DNA and RNA. The teratogenic and lethal effects of metabolically activated APAP observed in zebrafish embryos with our m DarT standard protocol could be explained by the lack of GSH as a detoxifying system. By adding GSH it was possible to mimic the situation in mammals and thus avoid teratogenic effects in zebrafish embryos.

Evaluation of a Procedure for the Simultaneous Quantification of 4-Ketocyclophosphamide, Cyclophosphamide, and Ifosfamide in Human Urine

An accurate and precise analysis procedure is presented for the detection and quantification of cyclophosphamide (CP), 4-ketocyclophosphamide (4-keto-CP), a primary metabolite of CP, and ifosfamide (IF) in human urine. CP and IF are common antineoplastic drugs used for the treatment of many types of cancer. Workers in the healthcare field, including nurses and pharmacists who interact with or prepare prescriptions for patients, have potential low-level exposure to the parent drugs; therefore, an analysis procedure is needed. The main focus of this procedure is the quantitation of 4-keto-CP because it is a primary metabolite of CP exposure and stable under physiological conditions. Sample preparation consists of liquid-liquid extraction of urine with ethyl acetate, and the analysis consists of reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry for detection of the analytes. Accuracy and precision of this procedure is demonstrated by means of recovery experiments. Recoveries are between 97-105% of theory for the three target analytes at various concentrations (25, 50, 100, and 375 ng/mL for 4-keto-CP; 1, 2, 4, and 15 ng/mL for CP and IF) with relative standard deviations of 8.4% or less. The limit of detection for this procedure is 1 ng/mL for 4-keto-CP, 0.1 ng/mL for CP, and 0.05 ng/mL for IF in urine.

Comet assay in murine bone-marrow cell line (FDC-P2)

The comet assay, also known as the single cell gel electrophoresis (SCGE) assay, is a rapid, simple, visual and sensitive technique for measuring DNA damage in mammalian cells. In the present study, Methyl methanesulfonate (MMS), 4-Nitrosoquinoline-Oxide (4NQO), Cyclophosphamide (CPA), and Benzo(a)pyrene (BP)-induced DNA damage was assayed in vitro in a murine bone-marrow cell line (FDC-P2), with or without an activation mixture (rat liver S9). All compounds caused significant DNA damage. With MMS and 4NQO, the frequency of comet tails, scored manually under a fluorescence microscope, increased dose-dependently, and reached a maximum of 53.2 and 74.8% respectively. Three parameters indicating DNA damage in the comet assay with the two-layer method, tail length, %DNA in tail, and tail moment, calculated using the automated image analysis software “Comet Analyzer v1.5” increased with all compounds. With MMS and 4NQO, all parameters increased at concentrations over 40 and 0.25 μmol/L, respectively. The in vitro comet assay with rat liver S9 could detect DNA damage caused by the metabolites of CPA and BP. The comet assay using the two-layer method is easy and efficient, and so can be conducted on a routine as basis. The assay with FDC-P2 cells was highly sensitive in detecting DNA damage with the frequency of comet tails, tail moment, %DNA in tail and tail length as indicators of the damage. Metabolism-mediated DNA damage could be detected with the addition of a rat S9 mixture at a final concentration of 6% for 6 h exposure.

Influence of cyclophosphamide and its metabolic products on the activity of peritoneal macrophages in mice

2,4,6-Trinitrophenyl (TNP) hapten-labeled peritoneal macrophages (Mf) given intravenously (iv) to recipients are poor inducers of contact sensitivity (CS) reactions unless Mf donors are pretreated with low doses of cyclophosphamide (CY). In vivo CY is converted into active alkylating metabolites, phosphoramide mustard (PM) and acrolein (ACR).Our experiments aimed to test how in vitro treatment of non-immunogenic Mf with different concentrations (10−5 to 10−7 M) of CY metabolites will influence their immunogenicity and other biological functions. Instead of chemically unstable PM, we used structurally and functionally similar nitrogen mustard (NM).Our experiments show that treatment of Mf with ACR or NM stimulates the in vitro production of pro-inflammatory IL-6 and IL-12 and down-regulates anti-inflammatory IL-10 and TGF-β cytokines. In vivo non-immunogenic TNP-Mf become capable of inducing CS reactions in two situations: first, after treatment with NM or ACR and second, when cell recipients are received iv before Mf transfer of monoclonal antibodies against IL-10 and/or TGF-β (500μg per animal). Treatment with NM, but not with ACR, was also an efficient stimulus for production by Mf of significantly increased levels of reactive oxygen intermediates (ROIs).In summary, our experiments show that CY metabolites can significantly increase the specific immune response as well as nonspecific innate reaction (ROIs production) and support the notion that CYand its metabolites can be a promising accessory tool when upregulation of the immune response is desired.

Dominant Effect of Antiangiogenesis in Combination Therapy Involving Cyclophosphamide and Axitinib

Antiangiogenic drug treatment inhibits tumor growth by decreasing blood supply, which can also reduce the delivery of other therapeutic agents. Presently, we investigated the effect of the vascular endothelial growth factor receptor tyrosine kinase inhibitor axitinib (AG-013736) on tumor vascular patency and chemotherapeutic drug uptake. Furthermore, the effect of axitinib on the antitumor activity of combination treatments with cyclophosphamide was examined. Prostate cancer PC-3 xenografts were used to evaluate the effect of axitinib treatment on tumor vascular morphology, fluorescent dye perfusion, hypoxia, and uptake of 4-hydroxycyclophosphamide, the active metabolite of the chemotherapeutic prodrug cyclophosphamide. Sequential or simultaneous schedules for axitinib and cyclophosphamide administration were evaluated in both PC-3 tumors and 9L gliosarcoma xenograft models. Axitinib monotherapy induced sustained growth stasis in PC-3 tumors in association with extensive apoptotic cell death. A substantial decrease in tumor vascular patency was observed, exemplified by a near complete loss of Hoechst 33342 perfusion and the absence of pimonidazole staining in the increasingly hypoxic tumors. Antitumor activity was significantly enhanced in both PC-3 and 9L tumors treated using an optimized schedule of sequential, intermittent axitinib-cyclophosphamide combination therapy despite a 40% to 70% decrease in tumor tissue uptake of 4-hydroxycyclophosphamide. In axitinib-cyclophosphamide combination therapy, enhanced anticancer activity can be achieved when the reduced tumor cell exposure to the cancer chemotherapeutic agent is compensated by antiangiogenesis-induced tumor cell starvation. This intrinsic antitumor effect was particularly evident in PC-3 tumor xenografts, where tumor blood flow deprivation dominates the overall therapeutic response.

Tempol reduces the therapeutic effect of cyclophosphamide on an experimental tumour model

The nitroxyl radical 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) is reported to elicite some effects on different biological models. This paper studied the influence of Tempol on the therapeutic action of an alkylating agent cyclophosphamide (CP) in the transplantable murine lymphosarcoma LS. When administered exactly before CP, Tempol exerted no influence on efficacy of the tumour therapy but significantly reduced it under a single or multiple preliminary injections 1–3 days before CP. This regimen of Tempol administration is found to elevate the activity of aldehyde dehydrogenase in the liver, the enzyme which is known to reduce the yield of the activated metabolite(s) of CP.

Environmental Stimulus Package: Potential for a Rising Oxidative Deficit

A close relationship exists between the multiple systems in the body and the gradual exposure to the environmental influences of oxidative stress, such as airborne particulate matter, pesticides, diet and lifestyle. Over time, exposure to exogenous sources of oxidative stress can ultimately impair both tissue and cellular function and lead to cellular senescence, apoptosis, and the destruction of nuclear chromatin. The effects of oxidative stress from the environment may be muted in some individuals if sufficient anti-oxidant defenses are in place. However, in others, comprised defenses such as those that can occur with immune system dysfunction, can lead to progressive damage in genetic coding and mitochondrial DNA that have been associated with signaling pathway errors and tumorigenesis. Interestingly, “multiple hits” by environmental toxins may be required to lead to system dysfunction or tumor growth. For example, a number of studies have observed that an accumulation of gene mutations with increased age may be necessary to eventually lead to cancers involving the breast or the gastrointestinal tract. Furthermore, the exposure to exogenous agents that may generate the release of reactive oxygen species also may initiate a critical cascade of cell injury. The occurrence of genetic mutations from the initial release of reactive oxygen species can then lead to polymorphisms in key enzymes such as glutathione S-transferase and cytochrome p450 that are vital to protect cells and tissues against oxidative stress. As a result of the loss of these anti-oxidant pathways, further accumulated tissue damage can ensue and the risk of developing significant illness increases further. Given the intimate relationship between the body and its susceptibility to exogenous sources of oxidative stress, we have highlighted a number of novel studies in this issue of Oxidative Medicine and Cellular Longevity that examine specific cellular and tissue responses to endogenous agents and the broad anti-oxidant systems that can mitigate injury during normal physiology and disease states. Kovacic and Somanathan set the stage for this issue with their opinion paper that describes the biology of a hallucinogenic agent that was an integral part of religious ceremonies by early North American natives and its use remains in our current society, namely mescaline. Their work provides new understanding for the cellular signaling of this agent in the nervous system, its role in structure function relations, and its ties to the hallucinogen psilocybin. In his review article, Flora broadens this perspective in regards to xenobiotics with metal and metalloid exposure. The biological properties of several agents that can lead to human illness are discussed that include copper, cobalt, and arsenic as well as the multiple anti-oxidant systems that can be activated, such as carotenoids, enzymes, hormones, phenolics, minerals, and vitamins. Interestingly, anti-oxidant systems also may play a significant role to prevent toxicity of chemotherapeutic agents. Al-Yahya et al. demonstrate that a naturally occurring branched-chain complex polysaccharide termed Gum Arabic can neutralize acrolein, a reactive metabolite of cyclophosphamide and block bladder toxicity from reactive oxygen species that may provide new direction for chemotherapeutic agents to enhance efficacy but limit toxicity. The ability to block cellular injury during periods of toxic elevated reactive oxygen species release also may be extended to a number systems throughout the body. Santo et al. show that ascorbic acid may be a powerful cytoprotectant in highly sensitive regions of the central nervous system during epilepsy and oxidative stress. Furthermore, in the paper by Mahapatra et al. that examines the effects of the exogenous toxin nicotine on immune cells, extracts from the plant Ocimum gratissimum were shown to have high flavonoid and phenolic content that may account for the ability of this agent to reduce super oxide anion generation, lipid peroxidation, and prevent cytotoxicity in immune cell peritoneal macrophages, suggesting that even the inflammatory effects of tobacco smoke that contains at least sixty known carcinogens may be reduced with appropriate anti-oxidant therapy. The efficacy of anti-oxidant therapy is brought to clinical consideration by Tripathi et al. in their paper. These investigators illustrate that administration of L-arginine, which has anti-apoptotic properties and can improve endothelial function, was able to lower serum cholesterol, increase superoxide dismutase function, and enhance the levels of total thiols in patients with cardiac ischemia, suggesting that chronic therapy with L-arginine in these patients may prevent the detrimental

17‐beta estradiol and prednisolone as potential stimulators of hair re‐growth in chemotherapy‐induced human hair follicle damage via ‘dystrophic catagen’ promotion?

Chemotherapy‐induced hair follicle (HF) damage and alopecia are common side‐effects of cancer therapy and constitute a major burden of disease in clinical oncology. Although several potential treatment strategies have shown promising effects in animal models, e.g. on cyclophosphamide‐induced alopecia (CIA) in C57BL/6 mice, these strategies remain to be transferred to human therapy. 17‐beta estradiol (E2) and glucocorticoids are potent modulators of murine CIA in vivo: topical treatment accelerates the regrowth of normally pigmented hair shafts by shifting HFs into the ‘dystrophic catagen’ pathway, which is associated with greater initial alopecia, but much faster HF recovery. In the current study, we asked whether E2 and/or prednisolon display similar activities in the human system, using our recently established human HF dystrophy in vitro‐model (Bodó et al., Am J Pathol 2007: 171(14):1153–67). E2 and/or prednisolon were pre‐ and co‐administered with a key cyclophosphamide metabolite (4‐HC). Indeed, E2 shifted 4‐HC‐treated human HFs into the dystrophic catagen pathway in vitro, and potentiated the 4‐HC‐induced pigmentary disturbances. 4‐HC‐induced hair growth inhibition was further enhanced by E2, associated with an additional reduction of proliferation and increased apoptosis of matrix keratinocytes. These effects tended to be further enhanced by co‐administration of prednisolon to the HF organ culture medium. These observations suggest that, just as in murine CIA in vivo, E2 and glucocorticoids can force chemotherapy‐damaged human HFs into the dystrophic catagen pathway. The preclinical data provide first indications that a combination of E2 with prednisolone may indeed serve as an effective clinical tool for accelerating hair re‐growth in human CIA.

The Gene Expression Profiles of Medulloblastoma Cell Lines Resistant to Preactivated Cyclophosphamide

The total expression profiles of two medulloblastoma cell lines resistant to the preactivated form of cyclophosphamide (4-hydroperoxycyclophosphamide, 4-HC) were examined using the Affymetrix GeneChip U133A array. Our primary objective was to look for possible genes, other than the well-studied aldehyde dehydrogenases (ALDH) that may be involved in cyclophosphamide (CP) resistance in medulloblastomas. We present here the lists of the most highly upregulated [30 for D341 MED (4-HCR); 20 for D283 MED (4-HCR)] and downregulated [19 for D341 MED (4-HCR); 15 for D283 MED (4-HCR)] genes which may be involved in conferring CP-resistance to the two medullobalstoma cell lines. The lists of genes from the two sublines almost had no overlap, suggesting different mechanisms of CP-resistance. One of the most noteworthy upregulated gene is TAP1 [90-fold increase in D341 MED (4-HCR) relative to D341 MED]. TAP1, a protein belonging to the ABC transporter family is normally involved in major histocompatibility class I (MHC I) antigen processing. This suggests the possible role of multidrug resistance (MDR), albeit atypical (which means it does not involve the usual MDR1 and MRP glycoproteins), in medulloblastoma's CP-resistance. Apart from TAP1, a number of other genes involved in MHC1 processing were upregulated in D341 MED (4HCR). D341 MED (4-HCR) also had a 20-fold increase in the expression of the aldo-keto reductase gene, AKR1B10, which may deactivate the reactive cyclophosphamide metabolite, aldophosphamide. For D283 MED (4-HCR), the most notable increase in expression is that of ALDH1B1, a member of the aldehyde dehydrogenase (ALDH) family of proteins.

The concept of neurogenic inflammation

Neurogenic inflammatory responses have recently been linked to both acute and chronic pathological conditions in the urinary tract. Neurogenic inflammation encompasses a series of vascular and non-vascular inflammatory responses, triggered by the activation of primary sensory neurons and the subsequent release of inflammatory neuropeptides, including substance P and calcitonin gene-related peptide. The reduction of neurogenic inflammatory responses may be key in the mode of action of the adrenergic alpha(1)-adrenoceptor antagonists used to treat lower urinary tract symptoms (LUTS). Indeed, the alpha(1)-adrenoceptor antagonist alfuzosin inhibits expression of the oncogene c-fos- a marker of nociceptive pathway activation - evoked by cyclophosphamide in rats. Capsaicin ameliorates urinary bladder symptoms through its stimulatory action on the transient receptor potential vanilloid 1 (TRPV1) calcium channel, resulting in desensitization of bladder sensory nerve terminals. Involvement of the TRP cation channel, subfamily A, member 1 (TRPA1) has also been reported in models of neurogenic inflammation and nociception promoted by the cyclophosphamide metabolite, acrolein. Blockade by alfuzosin demonstrates the beneficial effects of alpha(1)-adrenoceptor antagonists on neurogenic inflammation via the transient receptor potential family of ionic channels. Consequently, these drugs may have an important role in reducing LUTS.

Induction of COX-2 expression by acrolein in the rat model of hemorrhagic cystitis

Aim Acrolein (ACR) is a urinary metabolite of cyclophosphamide (CPS) and ifosfamide (IFS), which has been demonstrated to be the causative agent of hemorrhagic cystitis (HC), induced by these compounds. In this study, we investigate the participation of cyclooxygenase-2 (COX-2) on ACR-induced HC. Methods Male Wistar rats (150–200 g; six rats per group) were treated with distilled water or intravesical ACR and analyzed by changes in bladder wet weight, macroscopic and microscopic parameters and COX-2 expression. Results COX-2 immunohistochemical expression was significant 12 h after ACR administration mainly in subepithelial cells. ACR injection also alters some macroscopic and microscopic parameters in bladder of rats analyzed by Gray's criteria. Conclusions COX-2 participates in the pathogenesis of ACR-induced HC first seen 12 h after initial contact between ACR and urothelium.

Low-dose metronomic cyclophosphamide treatment mediates ischemia-dependent K-ras mutation in colorectal carcinoma xenografts

Antiangiogenic therapies are promising approaches to cancer control, but the details of their effects on subsequent tumor progression are not fully understood. Such therapies have the potential to eventually generate extensive amounts of tumor ischemia, and we previously demonstrated that ischemic conditions induce K-ras mutations in cells with deficient mismatch repair (MMR) mechanisms. This suggested that similar effects on oncogene mutagenesis may accompany antiangiogenic therapy. To test this, MMR-deficient colorectal cancer cells (Dks-8) were xenografted into immune-deficient mice and treated with the antiangiogenic regimen of low-dose/metronomic cyclophosphamide for 2 weeks followed by a 2-week recovery period without therapy. This treatment resulted in transient tumor growth inhibition, increased hypoxia, and decreased microvessel density, and cancer cells from treated tumors acquired activating mutations of the K-ras oncogene (K-ras(G13D)). In vitro exposure of Dks-8 cells to the active metabolite of cyclophosphamide (4-hydroxycyclophosphamide) had no effect on the K-ras status, indicating that there was no direct action of this alkylating agent on K-ras mutagenesis. In addition, cells sorted from hypoxic regions of Dks-8 tumors were enriched in K-ras(G13D) mutants. Collectively, our studies suggest that increases in tumor hypoxia induced by antiangiogenic treatment may lead to K-ras mutation and consequently tumor progression, especially in susceptible individuals.

Collaboration between hepatic and intratumoral prodrug activation in a P450 prodrug-activation gene therapy model for cancer treatment

Presently, we investigate the mechanisms whereby intratumoral expression of a cyclophosphamide-activating hepatic cytochrome P450 gene enhances therapeutic activity when cyclophosphamide is given on an every 6-day (metronomic) schedule. In P450-deficient 9L gliosarcomas grown in severe combined immunodeficient mice, metronomic cyclophosphamide substantially decreased tumor microvessel density and induced a approximately 70% loss of endothelial cells that began after the second cyclophosphamide treatment. These responses were accompanied by increased expression of the endogenous angiogenesis inhibitor thrombospondin-1 in tumor-associated host cells but by decreased expression in 9L tumor cells. These antiangiogenic responses preceded tumor regression and are likely key to the therapeutic activity of metronomic cyclophosphamide. Unexpectedly, 9L/2B11 tumors, grown from 9L cells infected with retrovirus encoding the cyclophosphamide-activating P450 2B11, exhibited antiangiogenic responses very similar to 9L tumors. This indicates that the tumor endothelial cell population is well exposed to liver-activated cyclophosphamide metabolites and that intratumoral P450 confers limited additional anti-endothelial cell bystander activity. In contrast, an increase in apoptosis, which preceded the antiangiogenic response, was substantially enhanced by intratumoral P450 2B11 expression. 9L/2B11 tumor regression was accompanied by an overall loss of tumor cellularity and by substantial enlargement of remaining P450-immunoreactive tumor cells as the number of P450-positive tumor cell decreased and the P450 protein content declined with cyclophosphamide treatment. We conclude that metronomic cyclophosphamide regresses P450-expressing tumors by two independent but complementary mechanisms: increased tumor cell killing via intratumoral P450-catalyzed prodrug activation, coupled with strong antiangiogenic activity, which is primarily associated with hepatic prodrug activation.

CHANGES IN MUSCARINIC RECEPTORS IN THE TOAD UROTHELIAL CELL LINE TBM‐54 FOLLOWING ACROLEIN TREATMENT

1. In cyclophosphamide-induced cystitis in the rat, cholinergic function of the bladder and muscarinic receptor expression are altered. In the present study, we investigated whether the toad urothelial cell line TBM-54 expresses functional muscarinic receptors and whether changes in muscarinic receptors can be induced in vitro by treating cells with acrolein, a metabolite of cyclophosphamide causing cystitis. 2. The occurrence of muscarinic receptors on cells was assessed by microphysiometry, a method analysing receptor function by measuring changes in the extracellular acidity rate (ECAR) in response to receptor stimulation. 3. Challenging untreated cells with the muscarinic receptor agonist carbachol gave rise to a concentration-dependent increase in changes in ECAR, with a maximal response at 1 mmol/L carbachol of 51 +/- 6%. Pre-incubating cells with different muscarinic receptor antagonists (i.e. pirenzepine (M(1) receptor selective), methoctramine (M(2)/M(4) receptor selective) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M(3)/M(1)/M(5) receptor selective)), gave rise to a concentration-dependent decrease in the effects of carbachol (0.5 mmol/L) on changes in ECAR. 4. Western blot analysis was used to determine the expression of all muscarinic receptor subtypes (M(1)-M(5)) by the cell line. Following acrolein treatment, cells were markedly less sensitive to carbachol and the expression of muscarinic M(2) receptors was decreased, whereas the expression of muscarinic M(3) receptors was increased. 5. In conclusion, the urothelial cell line TBM-54 expresses functional muscarinic receptors and exposure to acrolein leads to a modulation in the expression of muscarinic receptors. Consequently, acrolein may have direct effects on muscarinic receptor function and expression that contribute to the pathogenesis of cyclophosphamide-induced cystitis.

Dissecting the Impact of Chemotherapy on the Human Hair Follicle: A Pragmatic in Vitro Assay for Studying the Pathogenesis and Potential Management of Hair Follicle Dystrophy

Chemotherapy-induced alopecia represents one of the major unresolved problems of clinical oncology. The underlying molecular pathogenesis in humans is virtually unknown because of the lack of adequate research models. Therefore, we have explored whether microdissected, organ-cultured, human scalp hair follicles (HFs) in anagen VI can be exploited for dissecting and manipulating the impact of chemotherapy on human HFs. Here, we show that these organ-cultured HFs respond to a key cyclophosphamide metabolite, 4-hydroperoxycyclophosphamide (4-HC), in a manner that resembles chemotherapy-induced HF dystrophy as it occurs in vivo: namely, 4-HC induced melanin clumping and melanin incontinence, down-regulated keratinocyte proliferation, massively up-regulated apoptosis of hair matrix keratinocytes, prematurely induced catagen, and up-regulated p53. In addition, 4-HC induced DNA oxidation and the mitochondrial DNA common deletion. The organ culture system facilitated the identification of new molecular targets for chemotherapy-induced HF damage by microarray technology (eg, interleukin-8, fibroblast growth factor-18, and glypican 6). It was also used to explore candidate chemotherapy protectants, for which we used the cytoprotective cytokine keratinocyte growth factor as exemplary pilot agent. Thus, this novel system serves as a powerful yet pragmatic tool for dissecting and manipulating the impact of chemotherapy on the human HF.

Serious haematological toxicity of cyclophosphamide in relation to CYP2B6, GSTA1 and GSTP1 polymorphisms

Cyclophosphamide (CPA) is widely used as an anticancer and immunosuppressive agent in various indications, and the dosage given may vary considerably depending on the disease treated. A recent review [1] has described the pharmacokinetics of CPA, a prodrug of which 70–80% of the administered dose is activated by cytochrome P450 (CYP) to the active alkylating species 4-hydroxycyclophosphamide (4OH-CPA). Several CYPs are responsible for CPA activation, mainly CYP2B6 and, secondarily, CYP2C9, CYP2C19 and CYP3A4/5 (Figure 1) [2]. Systemic exposure to cyclophosphamide metabolites after fixed doses of cyclophosphamide may vary by up to 10-fold between patients [1]. Genetic variants of CYPs involved in CPA metabolism may contribute to its pharmacokinetic variability and genetic polymorphisms of glutathione S-transferases may influence its toxicity. Figure 1 Cyclophosphamide metabolic pathways (adapted from Timm et al.[2]). The mutations (1) CYP2B6516TT, (2) CYP2C9*1/*3, (3) CYP3A5*3/*3, (4) GSTA1*A/*B, and (5) GSTP1*A/*B were identified in the presented case We report a case of a patient who experienced severe toxicity (pancytopenia) with small dosages of CPA (1.60 mg kg−1 day−1). A 25-year-old female patient presenting with extramembranous glomerulonephritis (with tubulointerstitial lesions) began treatment with cyclophosphamide in association with prednisone (40 mg day−1). She also received the following treatments: an angiotensin-converting enzyme inhibitor, an oral anticoagulant, a statin and recombinant erythropoietin. When cyclophosphamide treatment was initiated, creatinine clearance was 32 ml min−1, leucocytes 6500 μl−1, absolute neutrophil count (ANC) 4550 μl−1, platelet count 345 000 μl−1 and haemoglobin 11.6 g dl−1. After 14 days of treatment (day 14), the patient was admitted to hospital because of an intense asthenia accompanied by fever (38.8–39.5°C) which had appeared 78 h previously. The patient had ulcerative pharyngitis with dysphagia, about 20 pyodermitis lesions infected by Staphylococcus epidermidis, total body hair loss and painful diarrhoea. Severe neutropenia (ANC < 100 μl−1) and anaemia (haemoglobin 8.8 g dl−1) were observed, leucocyte and platelet absolute counts were 6000 μl−1 and 178 000 μl−1, respectively. Hepatic enzymes were within normal range. All the treatments were stopped and antibiotherapy was initiated in association with an erythropoiesis-stimulating agent and recombinant granulocyte colony-stimulating factor. At day 20 the platelet count was 34 000 μl−1 and haemoglobin 5.8 g dl−1. At day 21, the patient was apyretic. Aplasia ceased on day 22. Pharyngitis and pyodermitis were cured on day 23 and hairiness was normal 3 months after cyclophosphamide discontinuation. As all the drugs previously used were recommenced without any side-effect and as the clinical symptoms observed caused a CPA overdosage, we therefore suspected an alteration of CPA metabolism or detoxification pathways. Genetic variants of enzymes involved in these pathways were explored. Results were the following: CYP2B6516TT, CYP2C9*1/*3, CYP2C19*1/*1, CYP3A4*1 A/*1 A, CYP3A5*3/*3, GSTA1*A/*B, GSTM1+ (presence of gene), GSTT1+, GSTM3*B/*B and GSTP1*A/*B. The major side-effects observed were aplasia and total body hair loss. They are more likely to be observed when cyclophosphamide is used as a cancer treatment (500–4000 mg m−2 day−1) than for nonmalignant kidney disorders (glomerulonephritis) (100–200 mg m−2 day−1). As leukopenia, thrombocytopenia and anaemia are dose-related, we hypothesized that the aplasia observed in the present patient, despite the low dose of CPA, could be explained by enhanced 4OH-CPA exposure because of increased activation pathway of CPA into 4OH-CPA and less active detoxification pathways of 4OH-CPA and phosphoramide mustard. This was specially as the CLCr was 32 ml min−1 and hepatic function was normal. Indeed, Xie et al.[3] have demonstrated in vivo (patients with haematological malignancies) that the CYP2B6516T variant allele contribution to CPA elimination clearance was about twice that from wild-type gene. Moreover GSTA1*B/*B polymorphism, corresponding to lower detoxification, was correlated with greater survival than GSTA1*A/*B or GSTA1*A/*A in breast cancer therapy with CPA [4]. The detoxification pathway of anticancer drugs was decreased in carriers of GSTP1 variant alleles [5]. Nakajima et al.[6] have demonstrated in vitro that inhibition of GSTP1 enhances the antitumour activity of CPA. The genetic polymorphisms observed in our patient might contribute to decreased detoxification pathways and to increased exposure to 4OH-CPA and could explain the reported adverse effects. Further studies are needed to confirm these results.

Role of MGMT in protecting against cyclophosphamide-induced toxicity in cells and animals

O 6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects cells from the biological consequences of alkylating agents by removing alkyl groups from the O 6-position of guanine. Cyclophosphamide and ifosfamide are oxazaphosphorines used clinically to treat a wide variety of cancers; however, the role of MGMT in recognizing DNA damage induced by these agents is unclear. In vitro evidence suggests that MGMT may protect against the urotoxic oxazaphosphorine metabolite, acrolein. Here, we demonstrate that Chinese hamster ovary cells transfected with MGMT are protected against cytotoxicity following treatment with chloroacetaldehyde (CAA), a neuro- and nephrotoxic metabolite of cyclophosphamide and ifosfamide. The mechanism by which MGMT recognizes damage induced by acrolein and CAA is unknown. CHO cells expressing a mutant form of MGMT (MGMT R128A), known to have >1000-fold less repair activity towards alkylated DNA while maintaining full active site transferase activity towards low molecular weight substrates, exhibited equivalent CAA- and acrolein-induced cytotoxicity to that of CHO cells transfected with plasmid control. These results imply that direct reaction of acrolein or CAA with the active site cysteine residue of MGMT, i.e. scavenging, is unlikely a mechanism to explain MGMT protection from CAA and acrolein-induced toxicity. In vivo, no difference was detected between Mgmt−/− and Mgmt+/+ mice in the lethal effects of cyclophosphamide. While MGMT may be important at the cellular level, mice deficient in MGMT are not significantly more susceptible to cyclophosphamide, acrolein or CAA. Thus, our data does not support targeting MGMT to improve oxazaphosphorine therapy.

In Vitro Effect of Fludarabine, Cyclophosphamide, and Cytosine Arabinoside on Chromosome Breakage in Fanconi Anemia Patients: Relevance to Stem Cell Transplantation

Designing stem cell transplantation (SCT) conditioning regimens for Fanconi anemia (FA) has proved difficult because of hypersensitivity to the DNA cross-linking agents. We performed chromosome fragility tests with 56 FA patients and with 50 non-FA patients with severe aplastic anemia or myelodysplastic syndrome. We evaluated peripheral blood lymphocyte specimens cultured for 72 hours and treated with mitomycin C, diepoxybutane (DEB), cyclophosphamide (CY) metabolites, cytosine arabinoside (Ara-C), and fludarabine (Flu) metabolite (9-beta-D-arabinofuranosyl-2-fluoroadenine [2-F-Ara-A]). The DEB and CY metabolite tests were highly sensitive and specific for FA (P<10(-4)) for both tests), and the number of aberrations per cell for DEB correlated with that for the CY metabolite test (P < 10(-4)) but did not correlate with the number of aberrations per cell for the Ara-C and 2-F-Ara-A tests. The difference in breakage frequencies between FA and non-FA patients for cultures treated with 2-F-Ara-A was not statistically significant. Most of the breakages observed in cells treated with 2-F-Ara-A-and Ara-C were chromatid breaks. It may be possible to determine the appropriate CY dose in the preconditioning regimen for SCT in FA patients on the basis of the in vitro effects on fragility, and Flu or Ara-C may be a safer drug than high-dose CY for conditioning in FA patients.

Glutathione S‐transferase A1 polymorphisms and acute graft‐vs.‐host disease in HLA‐matched sibling allogeneic hematopoietic stem cell transplantation

Busulfan and the metabolites of cyclophosphamide are conjugated with glutathione and catabolized by enzymes of the cytosolic glutathione S-transferases family. There are clearly linked single nucleotide polymorphisms in the promoter region of the glutathione S-transferase A1 gene (i.e., GSTA1*A, -567T, -69C and -52G; GSTA1*B, -567G, -69T and -52A). We assessed whether the clinical outcomes, including acute graft-vs.-host disease, of 61 patients with hematological malignancies, following HLA-matched sibling allogeneic stem cell transplantation using busulfan/cyclophosphamide conditioning are altered by glutathione S-transferase A1 genotypes. Globally, grade II-IV acute graft-vs.-host disease developed in 13 patients (21%). Grade II-IV acute graft-vs.-host disease developed in 15.2% of 46 patients with GSTA1*A/*A diplotype and in 40.0% of 15 patients with GSTA1*A/*B or GSTA1*B/*B diplotype (p = 0.04). Moreover, this relationship between GSTA1*A/*A diplotypes and lower incidence of acute graft-vs.-host disease was independent of the age, gender, stem cell source, and disease status. The incidences of acute skin graft-vs.-host disease were 7% (3/46) in patients with GSTA1*A/*A and 27% (4/15) in patients without GSTA1*A/*A (p = 0.009, univariate; p = 0.01, multivariate). Acute hepatic graft-vs.-host disease developed in 6 (13%) of 46 patients with the GSTA1*A/*A diplotype and in 4 (27%) of 15 patients without this diplotype (p = 0.09, univariate; p = 0.12, multivariate). Ten patients (16%) developed hepatic veno-occlusive disease. No significant difference was found in the incidence of hepatic veno-occlusive disease between patients with and without the GSTA1*A/*A diplotype (19.6% vs. 6.7%; p = 0.24). We conclude that the GSTA1*A/*A diplotype is an independent protective factor against acute graft-vs.-host disease, especially for skin graft-vs.-host disease, and probably for hepatic graft-vs.-host disease, in patients using busulfan/cyclophosphamide conditioning. The identification of glutathione S-transferase A1 genotypes prior to allogeneic stem cell transplantation could allow conditioning regimens and graft-vs.-host disease prophylaxis to be modified to improve outcome.

Mechanisms of Venoocclusive Disease Resulting From the Combination of Cyclophosphamide and Roxithromycin

High doses (>or=500 mg/m) of cyclophosphamide are known to cause venoocclusive disease (VOD). The authors recently observed a patient treated with immunosuppressive cyclophosphamide doses (100 mg/day) and roxithromycin who developed VOD. Because roxithromycin inhibits cytochrome P450 (CYP) 3A4 and P-glycoprotein, the patient may have been exposed to higher cyclophosphamide and/or cyclophosphamide metabolite concentrations. The effect of roxithromycin on the metabolism and toxicity of cyclophosphamide was studied using human hepatic microsomes and a human endothelial cell line. Cyclophosphamide or roxithromycin at concentrations from 0.05 to 500 micromol/L were not toxic to endothelial cells as assessed by lactate dehydrogenase (LDH) leakage assay. However, the combination of roxithromycin (500 micromol/L) and cyclophosphamide was toxic for all the tested cyclophosphamide concentrations (0.05 to 500 micromol/L) without clear concentration dependence (LDH ratio 38.3 +/- 11.0 [mean +/- SEM] for the combination with cyclophosphamide 0.05 micromol/L and 50.2 +/- 10.2 for the combination with cyclophosphamide 500 micromol/L; P <or= 0.005 for all tested combinations vs. control). Although roxithromycin did not favor the generation of toxic metabolites from cyclophosphamide, it led to cyclophosphamide accumulation due to inhibition of both CYP3A4 and CYP2B6. Although roxithromycin inhibited P-glycoprotein, this was not the mechanism by which cyclophosphamide toxicity was increased because cyclophosphamide in combination with other P-glycoprotein inhibitors was not toxic to endothelial cells. In the presence of roxithromycin (500 micromol/L), cyclophosphamide (500 micromol/L) induced apoptosis in endothelial cells (34.3 +/- 10.4% apoptotic cells [in % of total cells] for the combination of cyclophosphamide and roxithromycin, 0.7 +/- 0.25% for cyclophosphamide alone, 0% for roxithromycin alone; P < 0.0001) most probably by mitochondrial membrane permeability transition and release of cytochrome c. The combination cyclophosphamide and roxithromycin, but not the individual compounds, is toxic to endothelial cells by inducing apoptosis. Inhibition of P-glycoprotein and formation of toxic metabolites are unlikely causes.