Effect of disulfiram (tetraethylthiuram disulfide) amd diethyldithiocarbamate on the bladder toxicity and antitumor activity of cyclophosphamide in mice.

Cyclophosphamide is the most commonly prescribed alkylating agent in clinical medicine. The usefulness of cyclophosphamide is often limited, however, by its propensity to cause hemorrhagic cystitis especially in children or patients receiving concomitant radiotherapy. Administration i.p. of cyclophosphamide at doses of 100 mg/kg or more to mice produced a significant increase in urinary bladder weight within 48 hr of treatment. The present studies demonstrate that disulfiram prevented cyclophosphamide-induced bladder damage when administered p.o. within 1 hr of cyclophosphamide treatment. Diethyldithiocarbamate, a sulfhydryl-containing metabolite of disulfiram, had identical uroprotective activity. Unlike disulfiram, diethyldithiocarbamate was effective only when administered 2 to 4 hr after cyclophosphamide. Disulfiram augmented slightly the antitumor activity of cyclophosphamide against L1210 murine leukemia in vivo when administered 30 min prior to cyclophosphamide. In contrast, diethyldithiocarbamate had no effect on the antitumor activity of cyclophosphamide when administered 4 hr after cyclophosphamide.

Transfer of age-associated restrained tumor growth in mice by old-to-young bone marrow transplantation.

B16 melanoma and Lewis lung carcinoma grow more slowly in aged mice. Immunesenescent changes may account for this age-related difference. To test for the effect of immune deficiency on the growth of these tumors, we treated young mice with an immunosuppressive dose of radiation and then observed tumor growth. We also radiated young mice to a higher (lethal) dose and then rescued them with either young or old bone marrow transfusion. Tumors grew more slowly in radiated mice than controls and in those reconstituted with old bone marrow. These findings support the concept of immunesenescent-related reduced tumor growth.

Effects of a caspase and a calpain inhibitor on resting energy expenditures in normal and hypermetabolic rats: a pilot study.

Several diseases induce hypermetabolism, which is characterized by increases in resting energy expenditures (REE) and whole body protein loss. Exaggerated protein degradation is thought to be the driving force underlying this response. The effects of caspase and calpain inhibitors on REE in physiological and hypermetabolic conditions, however, are unknown. Thus, we studied whether MDL28170 (calpain inhibitor) or z-VAD-fmk (caspase inhibitor) affect REE under physiological conditions and during hypermetabolism post-burn. Rats were treated five times weekly and observed for 6 weeks. Treatment was started 2 h (early) or 48 h (late) after burn. In normal rats, MDL28170 transiently increased REE to 130 % of normal during week 2-4. z-VAD-fmk reduced REE by 20-25 % throughout the observation period. Within 14 days after burns, REE increased to 130+/-5 %. Whereas MDL28170/early treatment did not affect REE, MDL28170/late transiently increased REE to 180+/-10 % of normal by week 4 post-burn. In contrast, with z-VAD-fmk/early REE remained between 90-110 % of normal post-burn. z-VAD-fmk/late did not affect burn-induced increases in REE. These data suggest that caspase cascades contribute to the development of hypermetabolism and that burn-induced hypermetabolism can be pharmacologically modulated. Our data point towards caspase cascades as possible therapeutic targets to attenuate hypermetabolism after burns, and possibly in other catabolic disease processes.

Augmentations of glucose uptake and glucose transporter-1 in macrophages following thermal injury and sepsis in mice.

Glucose is the primary metabolic substrate of macrophages, which are critical components of the host response to injury and infection. We have carried out a series of studies to examine macrophage glucose uptake and the status of glucose transporter 1 (GLUT1) at both the mRNA and protein level. Peritoneal macrophages that were obtained from mice undergoing sham burned (S), 15%TBSA burn (B) +/- Pseudomonas aeruginosa burn infection (B + I) and lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) administration. [3H]deoxyglucose uptake was significantly increased (B, 157 +/- 9%; B + I, 243 +/- 19%; S + LPS, 231 +/- 24%; S + TNF-alpha, 379 +/- 18%; B + LPS, 230 +/- 13%; and B + TNF, 305 +/- 23%, P< 0.01 vs. sham). GLUT1 mRNA and protein levels were increased as well (mRNA: B, 135 +/- 13%; B + I, 250 +/- 33%; S + LPS, 282 +/- 29%; S + TNF-alpha, 193 +/- 19%; B + LPS, 378 +/- 20%; and B + TNF-alpha, 204 +/- 16%; protein: B, 159 +/- 27%; B + I, 181 +/- 17%; S + LPS, 219 +/- 26%; S + TNF-alpha, 343 +/- 51%; B + LPS, 366 +/- 41%; and B + TNF-alpha, 415 +/- 44, P< 0.01 vs. sham). Macrophages co-cultured with LPS or TNF-alpha in vitro demonstrated a similar response pattern. Following burn injury and infection, macrophages augment their cellular glucose uptake, which is facilitated by an increased GLUT1 mRNA and protein levels. TNF-alpha elicited by LPS may mediate this enhanced carbohydrate metabolism at the point of glucose entry into the cell.

Indomethacin augments granulocyte-macrophage colony-stimulating factor-induced hematopoiesis following 5-FU treatment.

We have previously reported that granulocyte-macrophage colony-stimulating factor (GM-CSF) given after the administration of 5-fluorouracil (5-FU) results in augmented hematopoietic recovery as evidenced by increased white blood cell and neutrophil counts. Mice receiving GM-CSF following 5-FU administration were observed to have a marked elevation in splenic granulocyte-macrophage colony-forming cells (GM-CFC) and a decrease in the femoral bone marrow GM-CFC. Because GM-CSF has been shown to increase prostaglandin synthesis and prostaglandins are thought to provide a negative feedback signal to down-regulate myelopoiesis, we sought to determine if the cyclooxygenase inhibitor, indomethacin, could prevent the reduction in the number of femoral bone marrow GM-CFC seen when GM-CSF was administered following 5-FU. Groups of mice received a single 60 mg/kg i.p. injection of 5-FU followed 24 h later by twice-daily injections of 1 micrograms GM-CSF and daily injections of 3, 5, or 6 mg/kg indomethacin; the hematopoietic assays were performed on day 7 following 5-FU. Compared to those animals that received GM-CSF alone following 5-FU, mice receiving 5 mg/kg indomethacin plus GM-CSF following 5-FU had increased numbers of GM-CFC in their bone marrow (3923 +/- 634 vs 971 +/- 138; p less than 0.001) as well as increased neutrophil counts (18,995 +/- 2872 vs 11,497 +/- 2476; p less than 0.01). Indomethacin alone was, in part, capable of facilitating hematopoietic recovery following 5-FU administration, but not to the extent seen when used in combination with GM-CSF. Prostaglandin inhibitors may have a role in combination with hematopoietic growth factors in accelerating hematopoietic recovery following cytoreductive chemotherapy.

Effect of ureteral ligation and nephrectomy on granulocyte-macrophage progenitor cells and azathioprine toxicity.

Using an in vitro quantitative clonal culture technique of bone marrow granulocyte-macrophage progenitor cells (colony-forming units culture (CFU-c)), we studied the hematopoietic toxicity of azathioprine after unilateral and bilateral ureteral ligation, unilateral and bilateral nephrectomy, and splenectomy in C57BL/6 mice. Analysis of femoral bone marrow 18 hr after i.p. injection of azathioprine (300 mg/m2) revealed increased CFU-c toxicity in comparison to controls as follows: (1) bilateral ureteral ligation, P less than 0.01; (2) bilateral nephrectomy, P less than 0.01; (3) unilateral ureteral ligation, P greater than 0.05 less than 0.1; (4) unilateral nephrectomy, P, not significant; and (5) splenectomy, P, not significant. Extrapolation from a dose-response curve for the toxicity of azathioprine on the bone marrow CFU-c indicated that bilateral ureteral ligation and bilateral nephrectomy had the effect of a 25 to 50% increase in the azathioprine dose. After bilateral ureteral ligation, serum granulocyte-macrophage colony-stimulating factor levels were increased and in vitro tritiated thymidine suicide studies showed an increased proliferative rate of the CFU-c. Since azathioprine is a predominantly cell cycle-specific agent, we suggest that increased sensitivity to azathioprine is related to the increased proliferative rate of the CFU-c. The findings provide a rationale for a clinical policy of azathioprine reduction when there is depressed renal function.

Hematopoietic toxicity by cimetidine. Reexamination using the antimetabolite azathioprine.

By using an in vitro quantitative clonal culture technique for bone marrow granulocyte/macrophage progenitor cells (GM-CFC), we studied the effects of cimetidine alone and cimetidine plus azathioprine on bone marrow toxicity in C57BL/6 mice. Femoral bone marrow showed no effect of cimetidine in doses from 31.25 to 500 mg/kg on either marrow cellularity or the number of GM-CFC. Cimetidine pretreatment with single doses of 62.5 mg/kg or 250 mg/kg had no effect on the bone marrow suppression of azathioprine at 100 mg/kg. Chronic cimetidine pretreatment at 62.5 mg/kg daily for 7 days also had no effects on the single-dose azathioprine toxicity. Cimetidine given either before or after azathioprine had no effect on the rate or final level of recovery of GM-CFC in the marrow after depletion by azathioprine. Cimetidine in vitro at doses of 3.1 to 200 micrograms/ml caused no alteration in the proliferative response of the GM-CFC. Analysis of the serum colony-stimulating activity 1 to 24 hr following doses of cimetidine of either 12.5 mg/kg or 31 mg/kg caused no change in the serum colony-stimulating activity. We could find no evidence that at clinically relevant doses, cimetidine increased the hematopoietic toxicity of the azathioprine or altered the rate of bone marrow recovery after azathioprine depletion.

Experimental tumors and aging: local factors that may account for the observed age advantage in the B16 murine melanoma model.

In the B16 murine melanoma model tumor growth has been shown to be slower in animals of advanced age. One feature associated with this slower growth has been prominent fibrosis demonstrated in biopsies of the tumor in older animals. We have performed experiments to examine the fibrotic response in young and old mice. In non-tumor bearing animals the capacity to regain skin strength after surgical laceration and healing by primary intention was greater in old mice. Histologic preparations suggested a more prominent fibrosis at the wound site. The animals who were injected subcutaneously with B16 melanoma and treated with L 3,4-dehydroproline (an inhibitor of collagen synthesis) local tumor growth was significantly enhanced only for the old animals. Although this inhibition of collagen synthesis produced a differential growth enhancement, there remained a significant difference in tumor volume between young and old animals. We conclude that fibrogenesis is an important host defense for containing local tumor growth and that this mechanism is preserved if not enhanced in mice of advanced age. Nevertheless other factors are needed to account completely for the observed age-advantage in the B16 melanoma model.

Systemic amyloidosis involving two renal transplants.

Two patients with primary amyloidosis, each of whom had received a renal transplant for chronic renal failure, developed amyloid in their allografts. In one patient amyloid was present primarily in glomeruli and to a lesser extent in the interstitial tissue. This patient developed renal failure necessitating retransplantation. In the second patient amyloid was seen in the interstitium and interlobular blood vessels. Minimal amyloid was present in the glomeruli. This patient died of cardiac amyloidosis with good graft function at the time of death. Of the several patients recorded in the literature with amyloid in renal allografts, our first patient is the only one to exhibit glomerular amyloid and failure of the graft. Amyloid in areas other than the glomerulus does not appear to be incompatible with satisfactory graft function.

Alterations of glucose transporter mRNA and protein levels in brain following thermal injury and sepsis in mice.

Since glucose transport and utilization are profoundly influenced by injury and infection, and the brain is an organ which primarily utilizes glucose as its energy source, we examined the status of the facilitative glucose transporters GLUT1 and GLUT3 in brain following thermal injury and infection. BDF1 mice underwent a 15% total body surface area burn with or without Pseudomonas aeruginosa infection. At 4 and 72 h post injury +/- infection, GLUT1 and GLUT3 mRNA abundance was measured by Northern blotting, and the correlative proteins determined using Western blotting. At 4 h, both brain GLUT1 mRNA and protein abundance were significantly increased in burned (mRNA 150 +/- 12%, protein 122 +/- 6%) and burn/infected (mRNA 165 +/- 11%, protein 119 +/- 5%) animals. At 72 h, GLUT1 mRNA and protein levels were also significantly increased in burn (mRNA: 139 +/- 11%, protein: 120 +/- 7%) and burn/infected (mRNA: 145 +/- 14%, protein: 138 +/- 8%) animals. Our studies suggest that alterations of GLUT1 mRNA and protein abundance were primary responses to the burn injury and were not further altered by burn wound infection.

Partial hepatectomy reduces the endotoxin-induced peak circulating level of tumor necrosis factor in rats.

The pro-inflammatory cytokine tumor necrosis factor (TNF) is dramatically and transiently elevated in the circulation during endotoxic and septic shock and is a primary mediator in the pathogenesis of the sepsis syndrome. TNF peaks in the circulation 90 min after endotoxin administration with little variation, even among species. The specific cells, tissues, or organs that produce the high circulating level of TNF in septic shock remain unknown. The most likely sources are macrophage-laden tissues such as the liver and the spleen and circulating blood leukocytes. This study evaluated whether the liver is an important source producing the TNF spike 90 min after endotoxin. To test this hypothesis, we measured the peak circulating level of TNF following an endotoxin injection in rats subjected to a two-thirds hepatectomy versus sham-operated controls. Hepatechectomized rats produced 64% less TNF after endotoxin than controls (857 + or - 143 pg/mL plasma vs. 2410 + or - 491, respectively; p < .01). In contrast, splenectomy did not significantly after peak TNF levels versus sham-operated controls following an endotoxin injection (1380 + or - 148 pg/mL plasma vs. 1710 + or - 291). Furthermore, incubation of rat blood with endotoxin for 90 min did not significantly increase TNF above controls. These experiments demonstrate an important role for the liver in producing the high circulating levels of TNF after an endotoxin injection and suggest that hepatic-specific cytokine modulation deserves study for a therapeutic benefit in septic shock.

Expression of the lactate transporter MCT1 in macrophages.

This study evaluated macrophage expression of the stereospecific lactate transporter, MCT1, and the effects of lipopolysaccharide (LPS), tumor necrosis factor (TNFa), or nitric oxide (NO) on MCT1 mRNA and protein levels and lactate transporter activity. Peritoneal and J774.1 macrophages were treated with either saline, LPS (10 or 100 ng/mL), dexamethasone (100 nM), and dexamethasone + LPS. Cells were harvested at 4, 8, and 16 h after treatment and processed for total RNA and protein isolation. LPS treatment significantly increased macrophage MCT1 mRNA expression at 4 and 8 h compared with the saline-treated cells. Dexamethasone did not alter MCT1 mRNA levels. Treatment of J774.1 macrophages with TNFalpha (1 ng/mL) or nitric oxide (DETA-NO, 100 microM) also significantly increased MCT1 mRNA levels at 4 and 8 h after treatment. LPS and TNFalpha treatment significantly increased MCT1 protein levels at 8 and 16 h after treatment. Lactate transporter activity in J774.1 macrophages was measured by uptake of 14C-labeled lactate. LPS and TNFalpha treatment significantly augmented lactate uptake, 12 h after administration; however, nitric oxide treatment did not affect lactate uptake. Thus, our data demonstrated that stimulated peritoneal and J774.1 macrophages express the lactate transporter, MCT, and that LPS and TNFalpha regulate MCT1 mRNA and protein levels.

Elevated expression of p47phox and p67phox proteins in neutrophils from burned rats.

The molecular control of neutrophil respiratory burst in burn injury was investigated through quantitation of protein factors, p47phox and p67phox, which are required for the activation of the phagocyte plasma membrane NADPH-oxidase. Circulating neutrophils were isolated from rats with 30% body surface area covered with full thickness burns. Neutrophil O2- generation, and p47phox and p67phox expressions, respectively, were determined using spectrophotometric and immunoblot techniques. Formylmethionyl-leucyl-phenylalanine stimulated superoxide anion generation was approximately 50% higher in neutrophils from rats 24 and 72 h after burns compared with that in sham control rats. The level of superoxide production was .47 +/- .05 nanomoles per minute per 10(6) cells (mean +/- SE, n = 6) at 24 h and .45 +/- .05 (n = 6) at 72 h postburn, whereas in sham control animals it was .32 +/- .02 (n = 8). Compared with the sham group p47phox levels, p47phox expression was 5.7-fold, 4.4-fold, and 4.5-fold higher, respectively, at 24, 36 and 72 h postburn. The levels of p67phox in burned animals were 2-fold higher than in the sham group, (p < .05) at 24 h postburn, and approximately 50% higher than sham at 36 h after the burn. The p67phox levels in rats 72 h after the burn were not significantly different from the sham values. These data support the occurrence of an up-regulation of p47phox and p67phox expressions accompanying the enhanced neutrophil respiratory burst activity during the early stages of burn injury. The up-regulation of p47phox and p67phox could be responsible for the priming of neutrophil O2- production leading to host tissue injury.

Effect of transfusion on oxygen transport in critically ill patients.

The role of isolated blood transfusion as a means toward improving oxygen transport was evaluated in 19 critically ill patients having sepsis syndrome as defined by standard criteria. ICU therapies were unchanged during transfusion and hemodynamic profiles with serum lactate levels were obtained before and after packed red blood cells were given. Blood transfusions in these patients did not cause a change in hemodynamic status. Arterial lactate determination was normal before and after transfusion was administered. Oxygen uptake failed to increase with transfusion, corresponding to increased arterial and mixed venous oxygen content. In the presence of sepsis, patients having oxygen delivery and uptake above normal without evidence of ischemia (normal lactate) will not increase oxygen consumption by raising the hemoglobin.

Molsidomine increases endotoxic survival and decreases cytokine production.

We hypothesized that nitric oxide (NO) may exert feedback regulatory control over cytokine production and improve survival in endotoxin (ETX) shock. To test this hypothesis, we evaluated the pre-endotoxin effect of the NO donor molsidomine (MOL) on circulating tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6 levels, the production of these cytokines in the perfused liver, and endotoxic lethality in mice. Male BDF mice weighing 28-32 g were administered either 100 mg/kg MOL or saline (SAL) i.p. Thirty minutes later, the mice received either 50 mg/kg Salmonella enteriditis ETX or SAL i.p. Mice were killed at 90 min after ETX or SAL, for either the determination of plasma cytokine levels by enzyme-linked immunosorbent assays or for use in the perfused liver assessment of cytokine production. MOL treatment significantly reduced the post-ETX circulating levels of TNF to 84%, IL-1 to 65%, and IL-6 to 56%, as compared with SAL-treated ETX controls. Endotoxic livers from MOL-pretreated mice produced 82% less TNF, 88% less IL-1, and 54% less IL-6 over a 2 h perfusion, as compared with SAL-treated ETX controls. MOL pretreatment also decreased lethality in ETX shock from 90 to 50% (p < .05). Therefore, NO may provide a beneficial effect during sepsis by inhibiting hepatic cytokine production, and thus providing survival benefits.

Role of neutrophils in burn-induced microvascular injury in the intestine.

The present study evaluated burn-induced vascular permeability alterations of rat small intestine in vivo and assessed the effect of neutrophil depletion in burn-injured rats on the altered intestinal microvascular permeability. 125I-labeled bovine serum albumin (125I-BSA) was injected intravenously, and its leakage from circulation into the intestinal tissue was determined by measuring tissue counts of 125I-BSA. Compared with sham, vascular albumin permeability increased 1.7-fold on day 1 post-burn and 3.0-fold on day 3 post-burn in ileum. In the jejunum, albumin permeability increased 1.8- and 2.5-fold on day 1 and day 3 post-burn, respectively. Intestinal tissue edema, determined as increases in tissue water contents, was noted in both intestinal segments on day 1 post-burn; no further increase in edema was found on day 3 post-burn. Neutrophil depletion before burn injury prevented the vascular leakage of albumin and edema in the ileum and jejunum on day 1 post-burn. On day 3 post-burn, the effect of prior neutrophil depletion on vascular permeability was less marked, and edema formation was not affected at all. These findings indicate that an absence of neutrophils prevents the loss of intestinal vascular barrier properties only in the initial periods after burns.

Glucocorticoid regulation of hepatic TNF production following cecal ligation and puncture sepsis.

Tumor necrosis factor-alpha (TNF) is a critical early mediator in the genesis of a systemic inflammatory response during a septic insult. Many of the harmful effects evident during sepsis are ascribed to excessive endogenous TNF production. Because the liver is an important source of circulating TNF during endotoxicosis, and because glucocorticoids are believed to have a regulatory role in suppressing endogenous TNF production, we evaluated the effect of adrenalectomy on the hepatic production of TNF in an isolated perfused liver model after cecal ligation and puncture (CLP) sepsis. Fasted, male Holtzman rats (n = 4/group) underwent CLP alone, adrenalectomy (ADREX) alone, or CLP plus ADREX (CLP/ADREX). Two hours after the operation, the rat livers were explanted and perfused in an isolated recirculating model. Serum TNF levels were greater in CLP/ADREX rats than in both other groups. TNF production in the perfused liver was greater in the CLP/ADREX rats when compared with either CLP alone or ADREX alone. A separate mortality study was performed (N = 35) that demonstrated a CLP induced mortality of 45%, and a CLP/ADREX mortality of 100%. Thus, adrenalectomy increased circulating TNF and hepatic TNF production as well as mortality in CLP sepsis. These findings suggest an important role for endogenous glucocorticoids in modulating hepatic TNF production during CLP-induced sepsis.

Enhanced expression of neutrophil NADPH oxidase components in intestine of rats after burn injury.

We have evaluated the accumulation of neutrophils in the gut and their infiltration into the intestinal extravascular spaces in rats subjected to a 25% total body surface area scald burn. The accumulation of neutrophils was assessed via measurements of myeloperoxidase (MPO) activity in the intestinal homogenates, and the immunohistochemical localization of neutrophil NADPH oxidase component proteins (p47phox and p67phox) within the intestinal extravascular spaces determined neutrophil tissue infiltration. MPO measurements demonstrated a 12- and 21-fold increase above the control value in the intestinal tissue at day 1 and day 3 post-burn, respectively, suggesting that a substantial total tissue accumulation of neutrophils occurs in the gut after burn injury. The immunohistochemical staining procedures showed both a definitive presence of the neutrophil in the intestinal extravascular spaces and an enhanced immunoreactivity in neutrophils accumulating in intestine after burn injury. There was no evidence of either the presence of neutrophils in the extravascular regions or any significant neutrophil immunoreactivity to NADPH oxidase component proteins in the intestines of sham control rats. These findings indicate that burn injury causes an enhanced migration of circulating neutrophils into the intestinal interstitial spaces and an upregulation of NADPH oxidase activity in the infiltrating neutrophils.

Is impaired wound healing caused by infection or nutritional depletion?

Since infection and malnutrition both impair wound repair, we examined the interactions of an intra-abdominal abscess and its nutritional alterations on wound healing. Mice subjected to a left paraspinal incision then underwent either cecal ligation and puncture (CLP) or sham operation (sham). Abscesses and an early decrease in food intake developed in all CLP mice, but the animals resumed normal food consumption by day 7. The CLP animals had an initial 20% weight loss; however, by day 11 they had a gradual return toward control weight. Fresh and formalin-fixed wound disruption strengths determined on days 7 through 35 revealed a significant weakness in CLP animals. To examine the role of nutritional intake, an additional set of experiments were performed with the following groups: CLP, sham, no operation (control), or sham operation with pair-feeding an amount equal to that consumed by the CLP group to reproduce only the nutritional deficit (sham/dep). At 7 and 11 days, fresh wound-disruption strengths of both CLP and sham/dep animals were significantly weaker than those of control or sham animals. A defect in formalin-fixed healing also existed for sham/dep mice at day 11. These findings suggest that short-term reduction in nutrient intake as a result of infection may be a major determinant of impaired wound healing.

Verapamil enhances antitumor activity without increasing myeloid toxicity.

The effectiveness of chemotherapy is limited by drugs' resistance and toxicity to normal host cells. Verapamil increases the cytotoxicity of the Vinca alkaloids and doxorubicin hydrochloride (Adriamycin) in tissue culture. In this experiment the effect of verapamil (VER) on the cytotoxicity of vincristine sulfate (VCR) and 5-fluorouracil (5-FU) was studied with the use of an intravenous lung colonization model. After receiving 5 X 10(4) B16 F10 cells intravenously, mice were randomized into six groups and treated with intraperitoneal injections of saline solution, VER, VCR, VCR plus VER, 5-FU, or 5-FU plus VER. In the first experiment, mice were killed on day 22 and lung colonies counted. In subsequent experiments, animals were monitored until death. The addition of VER to VCR significantly decreased pulmonary tumor formation (14 versus 47 colonies; p = 0.05). This was associated with an increase in mean survival from 40.4 to 53.7 days (p = 0.05). Although the addition of VER to 5-FU also decreased pulmonary tumor colony formation (26 versus 73 colonies; p = 0.001), there was no significant prolongation of survival with this treatment. A quantitative clonal culture of granulocyte-macrophage progenitor cells (GM-CFC) was used to assess the effect of VER on bone marrow toxicity. The addition of 5 mumol VER to 5-FU (5 X 10(-3) to 5 X 10(-7) mol/L) or VCR (5 X 10(-5) to 5 X 10(-9) mol/L) did not significantly reduce GM-CFC growth compared with treatment with either drug alone. In vivo marrow toxicity assessed 18 hours after a single drug injection was also not increased by the addition of VER to VCR or 5-FU. In this model, VER enhances the oncolytic effect of both VCR and 5-FU without a concomitant increase in toxicity to normal host marrow progenitor cells.