Search

Intensive cancer chemotherapy leads to significant bone loss, the underlying mechanism of which remains unclear. The objective of this study was to elucidate mechanisms for effect of the commonly used anti-metabolite methotrexate (MTX) on osteocytes and on general bone homeostasis. The current study in juvenile rats showed that MTX chemotherapy caused a 4.3-fold increase in the number of apoptotic osteocytes in tibial metaphysis, which was accompanied by a 1.8-fold increase in the number of tartrate-resistant acid phosphatase-positive bone resorbing osteoclasts, and a 35% loss of trabecular bone. This was associated with an increase in transcription of the osteoclastogenic cytokines IL-6 (10-fold) and IL-11 (2-fold). Moreover, the metaphyseal bone of MTX-treated animals exhibited a 37.6% increase in the total number of osteocytes, along with 4.9-fold higher expression of the DMP-1 transcript. In cultured osteocyte-like MLO-Y4 cells, MTX treatment significantly increased caspase-3-mediated apoptosis, which was accompanied by the formation of plasma membrane-born apoptotic bodies and an increase in IL-6 (24-fold) and IL-11 (29-fold) mRNA expression. Conditioned media derived from MTX-treated MLO-Y4 cells was twice as strong as untreated media in its capacity to induce osteoclast formation in primary bone marrow osteoclast precursors. Thus, our in vivo and in vitro data suggested that MTX-induced apoptosis of osteocytes caused higher recruitment of DMP-1 positive osteocytes and increased osteoclast formation, which could contribute towards the loss of bone homeostasis in vivo., (Copyright © 2011 Wiley Periodicals, Inc.)

Intensive use of cancer chemotherapy is increasingly linked with long-term skeletal side effects such as osteopenia, osteoporosis and fractures. However, cellular mechanisms by which chemotherapy affects bone integrity remain unclear. Methotrexate (MTX), used commonly as an anti-metabolite, is known to cause bone defects. To study the pathophysiology of MTX-induced bone loss, we examined effects on bone and marrow fat volume, population size and differentiation potential of bone marrow stromal cells (BMSC) in adult rats following chemotherapy for a short-term (five once-daily doses at 0.75 mg/kg) or a 6-week term (5 doses at 0.65 mg/kg + 9 days rest + 1.3 mg/kg twice weekly for 4 weeks). Histological analyses revealed that both acute and chronic MTX treatments caused a significant decrease in metaphyseal trabecular bone volume and an increase in marrow adipose mass. In the acute model, proliferation of BMSCs significantly decreased on days 3-9, and consistently the stromal progenitor cell population as assessed by CFU-F formation was significantly reduced on day 9. Ex vivo differentiation assays showed that while the osteogenic potential of isolated BMSCs was significantly reduced, their adipogenic capacity was markedly increased on day 9. Consistently, RT-PCR gene expression analyses showed osteogenic transcription factors Runx2 and Osterix (Osx) to be decreased but adipogenic genes PPARγ and FABP4 up-regulated on days 6 and 9 in the stromal population. These findings indicate that MTX chemotherapy reduces the bone marrow stromal progenitor cell population and induces a switch in differentiation potential towards adipogenesis at the expense of osteogenesis, resulting in osteopenia and marrow adiposity., (Copyright © 2011 Wiley Periodicals, Inc.)

The mucin Muc2 or Mycin2 (Muc2), which is the main structural component of the protective mucus layer, has shown to be upregulated during chemotherapy-induced mucositis. As Muc2 has shown to have protective capacities, upregulation of Muc2 may be a counter reaction of the intestine protecting against mucositis. Therefore, increasing Muc2 protein levels could be a therapeutic target in mucositis prevention or reduction. Our aim was to determine the role of Muc2 in chemotherapy-induced mucositis. Mucositis was induced in Muc2 knockout (Muc2(-/-)) and wild type (Muc2(+/+)) mice by injecting methotrexate (MTX). Animals were weighed and sacrificed on Days 2-6 after MTX treatment and jejunal segments were analyzed. Before MTX treatment, the small intestine of Muc2(+/+) and Muc2(-/-) mice were similar with respect to epithelial morphology and proliferation. Moreover, sucrase-isomaltase and trefoil factor-3 protein expression levels were comparable between Muc2(+/+) and Muc2(-/-) mice. Up to Day 3 after MTX treatment, percentages of weight-loss did not differ. Thereafter, Muc2(+/+) mice showed a trend towards regaining weight, whereas Muc2(-/-) mice continued to lose weight. Surprisingly, MTX-induced intestinal damage of Muc2(-/-) and Muc2(+/+) mice was comparable. Prior to MTX-injection, tumor necrosis factor-alpha and interleukin-10 mRNAs were upregulated in Muc2(-/-) mice, probably due to continuous exposure of the intestine to luminal antigens. Muc2 deficiency does not lead to an increase in chemotherapy-induced mucositis. A possible explanation is the mechanism by which Muc2 deficiency may trigger the immune system to release interleukin-10, an anti-inflammatory cytokine before MTX-treatment.

Nonhuman primates are of particular relevance in evaluating the potential toxicity of drugs and environmental agents. We have used previously published information and data from the present study to establish a relationship for New World (NW) and Old World (OW) primates on the basis of the frequency of spontaneous micronucleated erythrocytes (MNEs) observed in peripheral blood. Data on spontaneous MNEs in peripheral blood from 15 species of primates, including humans, indicate that NW primates have significantly (P < 0.01) higher MNE frequencies (group mean, 9.5 +/- 7.3 MNEs/10,000 erythrocytes; range, 0.7-20.5/10,000 erythrocytes) than OW primates (group mean, 1.0 +/- 0.9 MNEs/10,000 erythrocytes; range, 0.0-2.6 MNEs/10,000 erythrocytes). Humans are believed to have developed in the OW, and human MNE frequencies were similar to those described for OW primate species. We selected the common marmoset (Callithrix jacchus), a NW primate, to determine whether therapeutic pediatric doses of Metotrexate (MTX; 2.5 mg/kg), Cyclophosphamide (CP; 5 mg/kg), Cytosine-arabinoside (Ara-C; 3 mg/kg), or 5-Fluorouracil (5-FU; 10 mg/kg), administered daily for two consecutive days, increase the frequency of micronuclei. Micronucleated polychromatic erythrocyte frequencies were increased significantly in groups receiving MTX, CP and Ara-C, while MNE frequencies were increased by the Ara-C treatment. The results of this study indicate that NW primates have higher spontaneous MNE frequencies than OW primates, and because of this, NW primates like the common marmoset, may be suitable for evaluating the genotoxicity of chemical agents.

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure., (2004 Wiley-Liss, Inc.)

The detrimental effect of chronic administration of carbamazepine (CBZ) on serum and erythrocyte folates of patients has been extensively analyzed. However, at present, no data have been reported on the effect of CBZ on the intracellular content and activity of antimetabolite cytotoxic agents that can be used together with CBZ in the treatment of cancer patients. To investigate this issue, we chronically exposed in vitro the human ovarian cancer cell line SKOV-3 (grown under physiological folate concentrations) to CBZ, thus selecting SKOV-CBZ clones (SKOV-CBZ-50-2, SKOV-CBZ-50-5 and SKOV-CBZ-100-2) able to grow in the continuous presence of the antiepileptic drug. All of the SKOV-CBZ clones were more resistant to methotrexate (MTX) than the parental cells. MTX resistance index, as determined by the ratio between MTX concentrations inhibiting cell growth by 50% (MTX IC(50)) in SKOV-CBZ clones and in the parental cells, ranged between 3- and 5-fold. This resistance was related to a reduced intracellular content of MTX. No alteration activity of the cellular enzymes directly involved in MTX cytotoxicity (dihydrofolate reductase, thymidylate synthase [TS] and folylpolyglutamate synthetase) was observed. SKOV-CBZ clones were cross-resistant to the TS inhibitors tomudex and CB3717, but not to the TS inhibitor 5-fluoro-deoxy uridine and other antineoplastic drugs (cisplatin, doxorubicin, vincristine and taxol), whose cellular uptake is derived from transmembrane transport mechanisms different from folate receptor alpha (FRalpha) or reduced folate carrier (RFC). FRalpha mRNA and protein levels were significantly lower in SKOV-CBZ clones than in the parental cells. The reduced level of FRalpha in SKOV-CBZ clones was associated with a decreased binding capacity of folic acid. No variation of mRNA RFC expression in the SKOV-CBZ clones as compared to the parental cells was observed. Thus, after chronic exposure to CBZ, SKOV-CBZ clones develop resistance towards MTX due to defective MTX uptake., (Copyright 2000 Wiley-Liss, Inc.)

We have recently reported that albumin accumulates in solid tumors and serves there as a source of nitrogen and energy. Methotrexate-albumin conjugates [MTX(I)-RSA] derivatized at a molar ratio of 1:1 differ favorably from original MTX in terms of plasma presence and tumor uptake. The purpose of this study was to evaluate the therapeutic efficacy of these novel conjugates in a comparative study with low m.w. MTX is Sprague-Dawley rats bearing a Walker-256 carcinoma. The maximum tolerated dose (MTD) for MTX and MTX(I)-RSA was determined (2 mg/kg based on MTX injected on days 1, 3 and 8). The tumor-bearing rats received injections of either the MTD or MTD/2 of MTX, MTX-albumin or mixtures containing the MTD/2 or MTD/4 of both MTX and MTX-albumin. No toxic side effects were observed. Cure rate and tumor growth retardation were slightly better for the conjugate compared with MTX alone in the MTD group (16 complete remissions vs. 14 of 20 rats). The best results were achieved for the combination treatment with MTX and MTX-albumin, with complete remission in all 20 rats. In conclusion, MTX-albumin conjugates show therapeutic activity in vivo without toxic side effects. Additive effects were observed for a combination of MTX-albumin and MTX. These effects might be caused by the much longer tumor exposition time of the conjugate in conjunction with a different route of uptake (pinocytosis for MTX-albumin vs. folate receptors for MTX).

Methotrexate (MTX), a chemotherapeutic agent used to treat cancer, produces cytogenetic damage and has a cytostatic effect in a variety of test systems. Several antigenotoxic agents have been studied in various in vitro and in vivo systems. However, data are limited regarding their ability to modulate MTX-induced genotoxicity. In the present study, vanillin (VA) and chlorophyllin (CHL) were used as antigenotoxic agents to study their ability to minimize the DNA damage caused by MTX. Exponentially growing V79 Chinese hamster lung cells were treated with MTX at five different concentrations (5-100 micrograms/ml) with S9 activation for 6 h and post-treated with two concentrations of either VA (50 or 100 micrograms/ml) or CHL (50 or 100 micrograms/ml) for 40 h. Cytochalasin B was added for the micronucleus (MN) assay along with antigenotoxic agents to evaluate MN in binucleated cells. Chromosomal aberrations were also evaluated in parallel cultures. Results indicate that MTX alone induced a dose-dependent decrease in the nuclear division index (NDI) and the mitotic index (MI). A significant increase in percent micronucleated binucleated cells (MNBN) and percent aberrant cells (Abs) was observed. Studies using VA as an antigenotoxic agent showed a decrease in the number of MNBN (26.3-83.1%) and Abs (16.0-87.5%) with the addition of either 50 or 100 micrograms VA/ml. The addition of CHL also significantly reduced the number of MNBN (53.0-91.5%) at both concentrations tested. Chromosomal aberrations were also significantly reduced (41.0-83.0). These studies indicate that both VA and CHL are capable of effectively minimizing MTX-induced chromosomal damage.

A review of mishaps involving pediatric patients receiving anticancer chemotherapy was undertaken in order to assist intervention. Although the case literature is too sparse to provide definite recommendations, suggestions for management are made in the event of an error with a high risk (based on the case literature) of life-threatening toxicities. It is recommended that all incidents be reported in the literature in order to provide a basis for devising standard treatment protocols. It is also suggested that studies using animal models continue to be done in order to provide more experimental data about toxicities and potentially beneficial rescue therapies.

Cellular resistance to methotrexate (MTX) is believed to be unaffected by expression of MDR1 P-glycoprotein (Pgp), a pleiotropic efflux pump acting on different hydrophobic compounds that enter cells by passive diffusion. A series of human leukemic CCRF-CEM sublines, isolated by multi-step selection for very high resistance to MTX, exhibit multiple mechanisms of MTX resistance, including decreased carrier-mediated uptake of MTX and DHFR gene amplification. These sublines show cross-resistance to drugs of the multi-drug resistance (MDR) family, which is correlated with relative resistance to MTX. The MTX-selected sublines show increased expression and function of the MDR1 gene, based on the measurement of MDR1 mRNA, Pgp and rhodamine 123 accumulation. Sequence analysis of the MDR1 cDNA from MTX-selected CCRF-CEM cells revealed no mutations in the protein coding region. MTX resistance in these cell lines is partially reversible by a Pgp-specific monoclonal antibody (MAb) UIC2 and a monovalent FaB fragment of UIC2. Our results indicate that Pgp can contribute to multifactorial resistance to MTX.

Mortality is reported to be higher in methotrexate (MTX)-treated animals receiving total parenteral nutrition (TPN), compared to animals receiving enteral nutrition. The increased mortality is felt to be related to gut atrophy and bacterial translocation. In this study, we examined the effect of MTX (30 mg/kg) on survival, body weight loss, gut mass, and gut bacterial translocation in rats randomized to TPN or enteral nutrition. Twenty-four male Sprague-Dawley rats (n = 8 per group) were randomized to TPN, a peptide-based enteral diet (PEP), or CHOW. Animals were weighed daily and followed for survival (6 days). A separate group of rats (n = 6 per group) were similarly randomized and sacrificed at 3 days. Mesenteric lymph node complex, liver, spleen, lung, and blood were cultured for translocating bacteria. Body weight loss was similar in all groups (12.0-16.9 g/day). Mortality was significantly (P < 0.05) higher in the TPN animals (100%), compared to PEP (50%) and CHOW (25%) fed animals. All tissues in the TPN animals contained large quantities of bacteria, while most tissues in the CHOW group were free of bacteria. Bacterial counts in the PEP tissues were intermediate between TPN and CHOW. There were no significant differences between groups for gut weights or mucosal protein content. This study supports a direct relationship between bacterial translocation and mortality in rats following MTX.

Dihydrofolate reductase reduces folic acid to tetrahydrofolate as a prerequisite to one-carbon metabolism, which is required for normal embryonic de novo DNA synthesis. The developmental toxicity of methotrexate (MTX) has been attributed to MTX's ability to inhibit the activity of dihydrofolate reductase and thereby indirectly suppress one-carbon metabolism. The compound 1-(p-tosyl)-3,4,4-trimethylimidazolidine (TTI), which is structurally unrelated to folate, reestablishes one-carbon metabolism by the biomimetic transfer of single carbon units. Whether the developmental toxicity of MTX is indeed caused via suppressed one-carbon metabolism was tested in New Zealand white rabbits following concurrent maternal treatment with MTX and TTI. TTI reduced MTX developmental toxicity judged by increased mean fetal body weights, decreased percentage of malformed fetuses, and reduced incidences of major malformations. Two doses of TTI (90 mg/kg, each) at 1 hr prior to and 1 hr after MTX also reduced the developmental toxicity, but was no more effective than the single-injection regimen. Treatment with TTI alone caused no developmental toxicity. Histologically, MTX caused enlarged intercellular spaces in limb bud mesenchyme that began at 6-8 hr and increased in size until 16 hr. Mesenchymal nuclei appeared basophilic, with angular contours. Pretreatment with TTI delayed MTX-induced histological changes until 20-24 hr after MTX in 36-50% of embryos and completely protected the remainder. The sequence of MTX-induced changes was not altered among affected embryos, although the severity of the lesions did not appear as great. Saline-only or TTI-only treatments caused no alterations in limb buds. These data are consistent with the concept that impaired one-carbon metabolism is indeed the fundamental process underlying MTX developmental toxicity.

The hypothesis that valproic acid-induced dysmorphogenesis may be due to an interference of this drug with folate metabolic pathways was further investigated by a study of a possible interaction of valproic acid (VPA) and the established folate antagonist methotrexate (MTX). The dihydrofolate reductase inhibitor MTX (1.25 and 2.5 mg/kg, i.p.) was injected 15 min prior to VPA (300 and 400 mg/kg, s.c.) in day 8 pregnant NMRI mice. Fetuses were examined for exencephaly, resorption, and fetal weight retardation on day 18 of gestation. MTX produced no exencephaly or reduction in fetal weight, and the 2.5-mg/kg dose caused 56% resorption. Higher doses (5-20 mg/kg) produced embryolethality and fetal weight retardation, but no exencephaly. VPA (300 and 400 mg/kg) administration resulted in 3.4% and 12.6% exencephaly and 9% and 19% resorptions, respectively. Coadministration of MTX with VPA significantly increased VPA-induced resorption and exencephaly rates as well as fetal weight retardation. Exencephaly induced by VPA 400 mg/kg was increased to 29.5% and 24.1% (P < 0.01 and P < 0.05) when given with 1.25 and 2.5 mg/kg MTX, respectively. MTX (2.5 mg/kg i.p.) did not alter transplacental VPA (400 mg/kg, s.c.) pharmacokinetics. These results support the view that VPA-induced teratogenesis may be mediated by interaction with folate metabolism.

The contribution of the agents used in the CMF regimen, i.e., cyclophosphamide (CY), methotrexate (MTX) and fluorouracil (FUra), to the development of toxicity was determined in tumor-bearing WAG/Rij rats. Data from untreated (U) rats were compared with data from rats treated with single-agent therapy (C-, M- and F-treatment groups), with data from double-agent therapy (CM-, MF- and CF-treatment groups) and with data from the triple combination: the CMF-treatment group. Doses of agents of interest were the same in all treatment groups. The sequence of administration was (1) CY; (2) MTX and (3) FUra which is similar to clinical treatment with CMF. Systemic levels of CY, MTX and FUra were comparable to those found in patients treated according to the CMF regimen. Toxicity was evaluated by body-weight changes, water and food consumption, white blood cell (WBC) and platelet cell (Pts) counts. With the exception of WBC and Pts nadirs, estimated toxicity parameters reflected toxicity over the whole treatment period of 14 days. The toxicity was generally mild and well tolerated, with one fatality in the M-treatment group. CY was the main contributor to toxicity; it caused both myelotoxicity and gastro-intestinal toxicity. The contribution of FUra was judged to be negligible. MTX + FUra did not increase host toxicity in a synergistic or even an additional fashion. The absence of addition or synergism of toxic side-effects can be explained both by site-specific interactions at the pharmacodynamic level and by interactions at the pharmacokinetic level.

Methotrexate (MTX) is lethal or teratogenic to embryos of all species tested. New Zealand white rabbit embryos are relatively resistant to the embryolethal effects of MTX. However, when pregnant does were injected iv with 19.2 mg MTX/kg on gestational day 12, virtually all surviving fetuses exhibited multiple malformations of the head, limbs, and trunk. MTX is a structural analogue of folic acid that competitively inhibits dihydrofolate reductase, thereby preventing formation of folinic acid and essentially stopping one carbon metabolism. One carbon metabolism is important in the synthesis of methionine, histidine, glycine, and purine bases that are required for the de novo synthesis of DNA. Presumably these metabolic effects of MTX relate directly to its mechanism of developmental toxicity. An ameliorative treatment has been tested utilizing i.v. injection of pregnant rabbits with leucovorin (LV), a close structural analogue of folinic acid (the product of the inhibited enzyme), at various times after MTX exposure. When LV was injected at times up to 24 hours after MTX fewer malformed fetuses resulted and the incidence of specific malformations was reduced. When given at times up to 20 hours after MTX administration, LV virtually eliminated the grossly apparent effects of MTX at term. In the forelimb bud, MTX increased the extracellular space surrounding limb bud mesenchymal cells within 8-10 hours; this process continued through 16 hours and remained unabated by 24 hours. Mesenchymal cell nuclei became hyperchromatic and pyknotic during this time period. By 24 hours, a moderate amount of cellular debris was observed in the mesenchymal compartment of limb buds from approximately one-third of the embryos examined. Endothelial cell nuclei of the limb bud vasculature did not exhibit the histopathological alterations observed in the mesenchymal cells. Limb buds from embryos injected with LV at times up to 6 hours after MTX were histologically normal. When LV treatment was delayed until 16 or 20 hours after MTX, mesenchymal nuclei regained normal appearance within 2 hours of treatment; further, the abnormally large intracellular space began to decrease during the next 4 hours. Cellular debris was not a prominent feature of limb buds from LV-treated embryos examined at any time. Embryos from rabbits injected with LV at 24 hours after MTX exhibited either typical MTX-induced lesions or a sequence of reparative events similar to those described for the 16 and 20 hour LV-treated embryos.(ABSTRACT TRUNCATED AT 400 WORDS)

The available literature was searched for quantitative test results from both in vitro and in vivo assays for developmental toxicity for five model compounds: cyclophosphamide, methotrexate, hydroxyurea, caffeine, and ethylenethiourea. These compounds were chosen on the basis of their extensive utilization in a variety of assay systems for developmental toxicity as evidenced by their representation in the ETIC database (each generally has 100-500 citations encompassing multiple test systems). Nine cellular-based assays, six assays using whole embryos in culture, as well as Segment II and abbreviated exposure tests for mammalian test species are included in the database. For each assay, the critical endpoints were identified, each of which was then provided a three-letter code, and the criteria for extraction of quantitative information were established. The extracted information was placed into a computerized reference file and subsequently plotted such that the qualitative (positive/negative) and quantitative (e.g., IC50, highest ineffective dose (HID), lowest effective dose (LED] results across all test systems could be displayed. The information contained in these profiles can be used to compare qualitative and quantitative results across multiple assay systems, to identify data gaps in the literature, to evaluate the concordance of the assays, to calculate relative potencies, and to examine structure-activity relationships.

The folate antagonist methotrexate (MTX), widely used in chemotherapy, is a well-documented teratogen. However, the mechanism by which it exerts its effects is still unclear. Specifically, we have examined the cytotoxicity of MTX in vivo and in vitro and have looked at the relationship between cytotoxicity and teratogenesis. The chick embryo was utilized to examine the effects of the drug administered to carefully staged embryos. Embryos were exposed at stages 18-22 and examined on day 11 of incubation. Wings were malformed in a stage-dependent manner while legs were affected similarly at each stage used. A modification of the 51chromium-release assay was used to test the toxicity of MTX to limb cells in vitro. None of the tissues tested showed measurable toxicity in vitro even though the drug kills cells in vivo, thereby suggesting that MTX may be metabolized differently in vitro. Malformations induced by MTX do not seem to be due to changes in the amount of cell death taking place in the limb but may be caused by a transient inhibition of cell division.

Numerical chromosomal aberrations (aneuploidy) are a major factor in pregnancy wastage, birth defects, and mental retardation. Consequently, effective cytogenetic procedures in mammalian gamates are required for studying mechanisms and causes of chromosomal nondisjunction. Our aims were to determine for oocytes from superovulating prepubertal mice: (1) the yield of chromosomally scorable ova, following application of a double fixation procedure: (2) the background level of aneuploidy, and (3) the sensitivity to induction of aneuploidy (by methotrexate). Superovulation was induced in C129F1 hybrid mice, 22-24 days old, with pregnant mare serum and human chorionic gonadotropin (HCG). Diurnal photoperiodicity and injections were scheduled to assure HCG-induced ovulation of known timing. Methotrexate (200 mg/kg) was given at 3 hr and ova were recovered at 15 hr after HCG. We describe the adaptation of a double fixation procedure to mouse oocytes. Methotrexate led to significantly increased hypoploidy (2 1/2-fold) but not to the hyperploidy reported by others for adult mice. There was a high yield of ova with exactly countable chromosomes (average of 9.5 ova per mouse). Concomitantly, the background level of aneuploidy was very low (0/465 scorable ova were hyperploid). Given the additional advantages of economy and convenience, the superovulating 3-week-old mouse could be an effective source of ova for testing environmental agents for their aneuploidy-inducing potential; however, further studies are needed to establish the degree to which such ova are susceptible to aneuploidy induction.

The augmentation of methotrexate-induced embryotoxicity by aspirin was studied. Pregnant Charles River CD rats were given methotrexate or aspirin alone or in combination on gestation day 9 or 12. The frequency of fetal abnormalities was not affected and fetal body weight loss was not additive in the combined treatment. However, pretreatment with aspirin (200 mg/kg) significantly enhanced the embryolethality of methotrexate given at soes of 0.2 mg/kg on day 9 and 1.5 mg/kg on day 12. Studies with tritiated methotrexate in pregnant rats demonstrated that aspirin delayed the renal excretion of methotrexate and increased the concentrations of methotrexate in maternal plasma and the embryos. It is suggested that these effects are responsible for the observed potentiation of embryolethality.

High-dose methotrexate (HDMTX) with leucovorin (CF) "rescue" is being investigated for treatment of many malignant tumors. CF is usually begun 2 hours after ending the HDMTX infusion. However, since CF and methotrexate compete for the same cellular transport system, at high extracellular methotrexate concentrations it may be impossible to "rescue" cells with CF. A regimen of HDMTX with delayed leucovorin "rescue" was therefore designed. In this program, a 6-hour infusion of methotrexate (7.5 gm/m2) was followed 24 hours later by leucovorin "rescue." Nine patients with osteogenic sarcoma received 115 courses of this treatment. Toxicity was minimal. Plasma methotrexate values were identical to those following early CF 'rescue" regimens. HDMTX with delayed "rescue" is well tolerated. Although theoretically sound, further studies are needed to determine its efficacy in comparison to standard early "rescue" regimens.

Because of the frequent reports of hepatic toxicity associated with long-term administration of methotrexate, a rat model was developed utilizing daily methotrexate administration. This model revealed an incidence of fatty metamorphosis of over 80 percent, atrophy and necrosis of 30 percent, and fibrosis of 10 percent. Fatty liver changes did not differ substantially from control animals in those animals receiving long-term thydroxyurea, an agent which, like methotrexate, inhibits DNA synthesis but unlike methotrexate, does not impair methylation reactions. Because choline has a lipotropic effect and because its synthesis requires methylation, an attempt was made to block the liver toxicity of methotrexate by simultaneous administration of choline. Animals so treated did not show the pathologic changes in the liver characteristic of methotrexate treatment alone. Furthermore, the accumulation of triglycerides in the liver which was characteristic of methotrexate administration was markedly reduced in those animals receiving choline. These data strongly suggest that, in the rat model, methotrexate produced liver toxicity by virtue of an effect other than inhibition of DNA synthesis; and that this toxicity can be blocked without impairing methotrexate effect on bone marrow by the administration of choline, a lipotropic agent requiring methylation for its synthesis. It is suggested that these results may have implications for human therapeutic situations involving long-term administration of methotrexate.

A method was developed by which conjugates of methotrexate (MTX) with antibody were prepared via an oligopeptide spacer which, after internalization of the conjugates into the target cells, would be cleaved by lysosomal enzymes to liberate MTX or its derivative(s) for entry of the drug into the cytoplasm through the lysosomal membrane. The conjugate of MTX with a monoclonal antibody (MAb) (aMM46) to mouse mammary carcinoma MM46 cells prepared by this method via Leu-Ala-Leu-Ala showed potent, antibody-directed cytotoxicity through lysosomal degradation of the conjugate, most likely at the tetrapeptide spacer. This was supported by the following observations: (a) the cytotoxicity of the aMM46 conjugate was more potent than that of the corresponding normal gamma-globulin conjugate, the antibody alone not being cytotoxic; (b) the conjugate retained its potent cytotoxicity to MM46 cells even after removal, by hydroxylamine treatment, of a less stably bound MTX derivative which might have been released extracellularly and have caused non-specific cytotoxicity by entering the cells via the membrane active transport system for MTX; (c) the cytotoxicity was not inhibited by thiamine pyrophosphate, an inhibitor of the MTX transport system; (d) the cytotoxicity was inhibited significantly with ammonium chloride, which inactivates lysosomal enzymes by raising the pH; and (e) the cleavability of the Leu-Ala-Leu-Ala spacer by the lysosomal enzymes was verified by using bio-undegradable poly(D-lysine) and biodegradable poly(L-lysine) as the lysosomotropic macromolecular carriers: unlike the poly(L-lysine) counterpart, the direct MTX-poly(D-lysine) conjugate showed very weak cytotoxicity while the tetrapeptide-mediated conjugate of MTX with poly(D-lysine) exhibited potent cytotoxicity.

The prenatal effects of methotrexate (MTX) in rats and rabbits were assessed. It was found highly embryotoxic in postimplantation rat embryos; 0.3 mg/kg ip or less caused nearly total embryolethality with slight teratogenicity. Rabbit embryos were far more resistant to small doses of MTX than rats, but 19.2 mg/kg iv, when given during days 10 to 15 of gestation, produced little death and a constant spectrum of malformation in a high percentage of offspring. Cleft palate, skull defects, and severe fore- and hindlimb dysplasias, occurred with a high degree of regularity and were strongly dose and developmental-stage specific.

PCO, a yeast extract, offsets at least in part the mitotic inhibitory effect of methotrexate and fluorouracil on bone marrow cells in vitro but increases the antimitotic activity of the drugs on ascites Krebs-2 carcinoma under similar conditions. In vivo, PCO enhances the action of methotrexate against the L-1210 lymphoid leukemia and does not interfere with the effectiveness of fluorouracil against the ascites Krebs-2 tumor.

Studies were undertaken to investigate the relationship between cell-mediated and humoral immune responses in the rejection of L1210/MTX-Rev (LR) leukemia cells in CD2F1 mice. The anti-LR antibody (humoral) response was defined by both its cytotoxic antibody titer and isotype composition, assessed by a complement-dependent cytotoxicity assay and radioimmune assay, respectively. The cytotoxic thymus (T)-derived lymphocyte response in the spleen was quantitated by 125I release by 125I-IUdR-labelled target cells. Analysis of the sera of tumor-bearing mice indicated that the LR leukemia cells elicited a wide spectrum of anti-tumor antibody isotypes. In mice that mounted a rejection response, the IgM anti-LR response was transient, while in those that did not, the IgM response persisted. Antibody titers for all isotypes remained low until the onset of LR rejection. At that time, high titers of IgG anti-LR antibodies, predominantly of the IgG2a subclass, were detected in sera of tumor-free mice. Thus, the LR rejection response coincided best with the appearance of high titers of IgG2a anti-LR antibodies. A single i.p. injection of viable LR cells elicited a potent cell-mediated immune response; neither the appearance nor the magnitude of the cell-mediated immune response as measured in the spleen correlated well with the onset or the strength of the LR rejection response in the peritoneal cavity. The LR peritoneal cell population grew unabated in the presence of an intense spleen cell-mediated immune response, and the rejection process began at a time when little cellular immunity could be detected. These results suggest that in the rejection response IgM antibodies contribute little, if any, to the process, and the role of cytotoxic T lymphocytes is questionable (uncertain). The rejection of LR cells appears to be primarily mediated by IgG2a antibodies.

Intravenous high-dose methotrexate (HD-MTX) reduces cerebral glucose metabolism and produces behavioral abnormalities and electroencephalographic slowing in an animal model of acute HD-MTX neurotoxicity and in cancer patients undergoing HD-MTX chemotherapy. We used our model of HD-MTX neurotoxicity in the rat to determine if leucovorin (5-formyltetrahydrofolate) reduces this neurotoxicity, and extended our characterization of this model to identify regional as well as global HD-MTX treatment effects and to investigate HD-MTX-induced alterations in regional brain pH. Intravenous high-dose leucovorin reversed the HD-MTX-induced decrease in cerebral glucose metabolism and associated behavioral and electroencephalographic abnormalities in the rat, but low-dose leucovorin was ineffective. The major effect of HD-MTX on cerebral glucose metabolism was a global reduction; however, smaller region-specific treatment effects were identified in auditory, thalamic, and white matter structures. HD-MTX did not alter regional brain pH. These findings suggest a potential clinical role for high-dose leucovorin in severe or prolonged acute HD-MTX neurotoxicity and provide an important justification for the role of positron emission tomography in the early detection of clinical HD-MTX neurotoxicity.

The relationship between polyene macrolide-induced early membrane damage and cytotoxicity in B1 (hamster), B82 (mouse), and RAG (mouse) cells has been investigated. Filipin (FIL) induced the greatest immediate damage, as monitored by 51Cr release, followed by mediocidin (MED), amphotericin B-deoxycholate (Fungizone) (FZ) and pimaricin (PIM). For long term effect, PIM was the least toxic followed by MED, FZ, and FIL as indicated by 24-hour survival, 72-hour viability, and growth rate of cells. In evaluating polyene macrolide-induced permeability alterations and cytotoxicity two types of interactions with mammalian cells were found: (1) cell toxicity at polyene macrolide levels not eliciting immediate membrane permeability changes; and (2) immediate membrane damage without long range toxicity.

An in vitro culture of chicken embryo is described: the embryos at the stage of gastrulation are explanted from eggs into transparent silicone chambers where they continue to develop normally under controlled conditions for additional 3 d. This period corresponds to 2-5 wk of postconceptional age in the human embryo. In both the chick and man, this period is very sensitive to physicochemical perturbations which can lead to surviving malformations. Six chemical agents were tested with this culture: methotrexate, cadmium chloride, caffeine, phenobarbital, aspirin, and saccharin. Survival scores, growth perturbations, and early signs of anomalies of the nervous, skeletomotor, and cardiovascular systems were analyzed with respect to the used concentrations. The dose-response curves were obtained with good precision and allowed a discrimination between the teratogenetic and unspecific toxic effects and a comparison of the toxic potency of the six drugs. The evaluation of one drug took, roughly, 3 wk, one technician, and about 150 eggs. The advantages (simplicity, rapidity, reproducibility, specificity, economy, no suffering, and no use of mature animals) and disadvantages (nonmammalian species, absence of detoxicating organs) of the method are discussed. The method is proposed as a routine teratogenicity and embryotoxicity test which allows primary screening of many compounds and which can thus substantially reduce the ultimate experiments that use pregnant mammalian females.

Intravenous high-dose methotrexate chemotherapy may produce acute, subacute, or chronic neurotoxicity in patients with cancer. Acute encephalopathies following high-dose methotrexate treatment are recognized with increasing frequency. This study describes a model of acute high-dose methotrexate neurotoxicity in the rat characterized by a profound dose-dependent depression of cerebral glucose metabolism in association with behavioral and electroencephalographic abnormalities. Alterations in the amino acid profile, similar to those described in cancer patients after high-dose methotrexate treatment, were observed in the absence of biochemical evidence of systemic organ toxicity. This model facilitates the study of the biochemical mechanisms of antifolate neurotoxicity in humans and permits the evaluation of potential therapeutic interventions.

The unexpected discovery that certain chemotherapeutic agents used in the treatment of childhood cancers have neurocognitive side effects has prompted a search for techniques that identify those medications that place children at risk. An animal model for the assessment of resultant neurocognitive toxicity is described which makes use of simple classical conditioning. We have shown that rats learn about environmental events more slowly following neonatal administration of methotrexate. The changes after methotrexate exposure are not related to stimulus characteristics or to perceptual abilities, but rather to damage to the neural systems involved in acquisition, retention, or recall. Similar problems with learning have been observed in children treated with methotrexate. An effective animal model such as the one described here may help detect and avoid antineoplastic agents that produce severe cognitive defects in childhood cancer patients.

Methotrexate (MTX), administered as a single 20 mg/kg intraperitoneal dose to C57Bl/6J mice on their 9th day of pregnancy results in high incidences of median facial clefts in the surviving gestational-day-18 fetuses. We have shown the presence of dilated and congested blood vessels in the frontonasal prominences (FNP) of embryos from treated mothers as early as 3 hours following drug administration. Within 24 hours, large vascular blebs are located in the FNP and the neural tubes appear somewhat distended. By 32 hours after treatment, distention of the neural tube is marked while blebs have become less evident. Subsequent to these changes, FNP mesenchymal deficiency as well as neural tube distention lead to the formation of median facial clefts. It is hypothesized that, as with a number of other teratogenic agents (especially hypoxia), initial fluid imbalance is the primary teratogenic insult.