Divisions of 1 Clinical Pathology, 3 Oncosurgery and 6 Digestive Surgery, University Hospital, Geneva, Switzerland; Services 2 dAnatomie Pathologique, 4 Gastroenterology and 5 Surgery, Hôpital Cochin, Paris, France
Received 15 July 2003; revised 29 September 2003; accepted 16 December 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In advanced metastatic colorectal adenocarcinoma, the addition of a neo-adjuvant systemic treatment to surgery might translate into a survival advantage, although this is yet to be confirmed by ongoing randomized trials. The objective of this study was to assess the effects of preoperative systemic chemotherapy on the morphology of non-tumoral liver.
Patients and methods:
A large series of surgically resected liver metastases (n = 153) was selected. Light microscopy, electron microscopy, and immunohistochemistry using antibodies against endothelial cells (CD31) and hepatic stellate cells (-SM actin, CRBP-1) were performed to identify sinusoidal wall integrity.
Results:
We found that 44 (51%) of the 87 post-chemotherapic liver resection specimens had sinusoidal dilatation and hemorrhage, related to rupture of the sinusoidal barrier. In contrast, the 66 livers treated by surgery alone remained normal. In 21 out of the 44 post-chemotherapy patients (48%), perisinusoidal and veno-occlusive fibrosis also developed. Sinusoidal injury persisted several months after end of chemotherapy, and fibrosis may progress. Development of lesions was strongly correlated to the use of oxaliplatin; 34 out of 43 patients (78%) treated with this drug showed striking sinusoidal alterations.
Conclusions:
Systemic neo-adjuvant chemotherapy in metastatic colorectal cancer frequently causes morphological lesions involving hepatic microvasculature. Sinusoidal obstruction, complicated by perisinusoidal fibrosis and veno-occlusive lesion of the non-tumoral liver revealed by this study, should be included in the list of the adverse side-effects of colorectal systemic chemotherapy, in particular related to the use of oxaliplatin.
Key words: drug liver injury, 5-fluorouracil, neo-adjuvant chemotherapy, sinusoidal obstruction syndrome, veno-occlusive disease
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Increasing numbers of liver metastases are now considered surgically resectable. Various factors explain this trend: progress of surgical techniques, better knowledge of the functional anatomy of the liver, detection of metastases at an earlier stage, introduction of new therapeutic approaches (e.g. cryosurgery, radio- frequency) and use of chemotherapy before resection. This latter approach, aimed at the reduction of the size of metastases, allows the resection of an additional 16% of metastases previously deemed unresectable [4].
Despite the large number of drugs available, only a few agents have been found to be active in advanced colorectal cancer. The standard treatment for advanced colorectal cancer has been 5-fluorouracil (5-FU)-based chemotherapy combined or not with leucovorin. Recently, new therapeutic approaches have been introduced. These include agents with mechanisms of action unrelated to thymidylate synthetase, such as irinotecan, a topoisomerase I inhibitor [5], and oxaliplatin, the only platinum derivative with significant activity in colorectal cancer [6]. Combined with 5-FU and leucovorin, both irinotecan and oxaliplatin have led to a significant improvement in terms of response rate, disease-free and even overall survival in metastatic disease, and have become standard of care in this setting. Considerable enthusiasm for the use of these new systemic combinations in a neo-adjuvant setting before curative surgical removal of liver metastases has also generated a large set of very encouraging data, but only in the context of non-controlled trials, and confirmation in proper randomized trials is still awaited [711].
Despite the increasingly extensive use of neo-adjuvant chemotherapy in advanced colorectal cancer, its functional and histopathological effects on the liver remain inadequately described. The aim of this study was to attempt to fill this gap by analyzing the morphological effects of preoperative systemic chemotherapy on the non-tumoral hepatic parenchyma, in a large series of livers resected for metastatic involvement by colon carcinoma. The study was conducted in two centers that have used preoperative chemotherapy since 1995.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The group included 93 men and 60 women. The mean age was 62 years (range 3680). The liver resections consisted of 80 right and 31 left hepatectomies, and 42 segment-oriented resections. Vascular exclusion (Pringles maneuver or vascular efferent exclusion) was performed during surgery in 55 cases. Preoperative portal embolization was performed in six cases. Eighteen patients underwent two or more liver resections, because of recurrence of hepatic metastases in 16 cases, and two time resections in two cases.
Surgical resection was the only treatment undergone for the metastases in 66 cases. Systemic chemotherapy was undergone before hepatic surgery in 87 cases. Several different protocols were used: 16 patients received oxaliplatin in combination with irinotecan and 5-FU; 27 received oxaliplatin in combination with 5-FU; 17 received irinotecan in combination with 5-FU; and 27 received 5-FU alone (Table 1). The administration was exclusively intravenous.
|
The following histological features were evaluated: sinusoidal dilatation, centrilobular vein fibrosis, perisinusoidal fibrosis, nodular hepatic regeneration, hepatocellular necrosis and steatosis. Sinusoidal dilatation was graded semi-quantitatively as follows: 0, absent; 1, mild (centrilobular involvement limited to one-third of the lobular surface); 2, moderate (centrilobular involvement extending in two-thirds of the lobular surface); 3, severe (complete lobular involvement). Steatosis was estimated as the percentage of involved hepatocytes, and was categorized as follows: 0, absent; 1, mild (steatosis in <30% of the hepatocytes); 2, moderate (steatosis in 3060% of the hepatocytes); 3, severe (steatosis in >60% of the hepatocytes).
The 2-test was used to correlate the different groups of patients according to the presence or not of preoperative treatment, and the type of chemotherapy versus the presence of sinusoidal dilatation. A result was considered statistically significant if P <0.05.
Immunostaining
Five cases of hepatectomies showing representative sinusoidal lesions and five control cases of hepatectomies for colorectal metastasis without lesions were selected for immunohistochemical studies, in order to better characterize the morphology of the sinusoidal lesions.
For immunostaining, additional serial 35 µm sections were prepared. Sections were mounted on silane-coated glass slides, air-dried overnight, deparaffinized, rehydrated and pretreated with H2O2/methanol to block endogenous peroxidase activity. The following primary antibodies were used: mouse monoclonal -smooth muscle (
-SM) actin (DakoCytomation, Zug, Switzerland; 1:400 dilution); rabbit polyclonal cellular retinol binding protein-1 (CRBP-1) (gift from Professor G. Gabbiani; 1:200 dilution); and mouse monoclonal anti-human CD31 (DakoCytomation; 1:50 dilution). For
-SM actin and CD31 antibodies, microwave pretreatment was used. For CRBP-1, pretreatment with a pressure cooker was performed. Sections were incubated for 1 h at room temperature with the diluted primary antibodies, which were then revealed by ENVISION (DakoCytomation). Peroxidase activity was revealed with 30% 3,3'-diaminobenzidine as chromogen in phosphate-buffered saline containing 0.015% H2O2. Sections were weakly counterstained with Mayers hematoxylin and mounted in Eukitt (Kindler GmbH, Freiburg, Germany). Negative controls were prepared by omitting the first antibodies.
Electron microscopy
During the study period, new available liver specimens with macroscopic lesions were fixed in glutaraldehyde and embedded in Epon. Semi-thin sections were stained with Toluidine Blue. Regions of the liver with representative sinusoidal lesion were selected for thin sections. Thin sections were treated with uranyl acetate and lead citrate and examined using a Zeiss electron microscope (EM CR 10; Zeiss).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
In livers with sinusoidal lesions, CD31 positivity was absent from areas of severe sinusoidal dilatation. In contrast, CRBP-1 showed the same distribution as observed in normal liver (data not shown). These results suggest loss of sinusoidal endothelial cells but a persistence of perisinusoidal HSC along dilated sinusoid. However, the expression of -SM actin in HSC was drastically modified compared with normal liver. A strong reactivity was present in almost all HSC within the lobules, and was present both along dilated sinusoids and intact sinusoids (data not shown). This indicates an activated state of HSC.
Electron microscopy
Extensive ultrastructural abnormalities of sinusoidal endothelial cells were observed. Endothelial cells were rounded up and swollen; their nuclei showed peripheral chromatin condensation, suggesting the apoptotic process (Figure 2A). Areas of sinusoidal dilatation showed diffuse discontinuities along the endothelial sinusoidal lining, allowing red blood cell extravasation in the widened Disses spaces (Figure 2A and B). A neodeposition of extracellular matrix components in Disses spaces was evident, mainly by fine collagen bundles (Figure 2A). A striking feature was the presence of numerous cytoplasmic blebs obstructing the sinusoidal lumen, several of them containing organelles (Figure 2B). In contrast, hepatocytes were better preserved, showing mainly a loss of hepatocyte microvilli.
Correlation of histological findings with type of chemotherapy and clinical data
Centrilobular lesions were observed in 44 of the 87 (51%) patients who received chemotherapy. No centrilobular lesions were observed in any of the 66 patients treated by surgery alone, including those in whom preoperative portal embolization or preoperative vascular exclusion had been performed (Table 1). Among the different protocols of chemotherapy, a significant correlation was detected between the presence of liver lesions and the use of oxaliplatin: 34 of the 43 (79%) patients treated with oxaliplatin developed lesions, as opposed to 10 of the 44 (23%) who did not (P <0.001) (Table 1). The amount of oxaliplatin received by our population was quantified as cumulative dose expressed in mg/m2; the range was 2801600 mg/m2. No correlation was found between the cumulative dose of oxaliplatin and the development of sinusoidal lesions (P >0.05).
The mean interval between last administration of chemotherapy and surgical resection was 35 days (range 16110). Hepatic sinusoidal lesions persisted up to 4 months after last chemotherapy. Steatosis was observed in 42 of the cases 87 (48%) with chemotherapy and in 33 of the 66 cases (50%) without chemotherapy (P >0.01).
Evolution of the liver lesions
To appreciate the evolution of the liver lesions at a time distant from the initial chemotherapy, we analyzed the 18 cases who underwent iterative hepatic surgery. In three patients treated by surgery alone, no centrilobular lesions were observed in the subsequent hepatectomies. Among the 15 remaining patients, nine had initial lesions on the first hepatectomy. Six of them received new cycles of preoperative chemotherapy, which did not allow the evaluation of the natural evolution of the lesions at a time distant from the end of chemotherapy. The three remaining patients showing lesions on the first liver resection had iterative hepatic resections for recurrence of liver metastasis with surgery as the only treatment. The second hepatectomies were performed 5, 11 and 17 months after the first resection, respectively. A third hepatectomy was performed in one patient 32 months after the second one. There was a persistence of sinusoidal dilatation in one case and fibrosis in two cases. In one of these patients, the fibrosis progressed; it no longer remained confined to the centrilobular zones, but it also extended to the rest of the lobule with centro-centro and porto-centro bridging fibrosis, reaching a cirrhotic configuration in the third hepatectomy (Figure 1D). In this patient, -SM actin-positive HSC were diffusely present along the lobules, while the pattern of CRBP-1 and CD31 expression was similar to that found in normal liver (data not shown). Moreover, this patient was hepatitis B and C virus negative, and had no history of alcohol abuse.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of this study show that neo-adjuvant chemotherapy in patients with metastatic colon cancer may induce important sinusoidal obstruction associated with sinusoidal fibrosis and veno-occlusive lesions, occasionally with nodular regenerative hyperplasia. In our population, this effect was particularly marked in the livers of patients treated with protocols that included oxaliplatin. No such lesions were present in the resected livers from patients who had been treated by surgery alone.
Whereas hepatocellular carcinomas usually arise from a background of fibrosis or cirrhosis, colon cancer metastases often affect normal livers. Frequently in our experience, however, surgeons report gross abnormalities, such as soft, congestive or even pseudocirrhotic livers, which compel them to limit the extension of the resection. Not uncommonly, frozen-section examinations requested to evaluate the non-tumoral liver prior to the performance of the resection indicate sinusoidal lesions.
To our knowledge, these abnormalities have not been systematically investigated. Only the hepatotoxic effects of 5-FU have received much attention: Washington et al. [13] reported in 1993 the development of NRH in five patients treated with 5-FU; bile duct fibrosis and stenosis secondary to hepatic intra-arterial 5-FU treatment have also been reported [14]. Furthermore, experimental studies with the use of intra-arterial 5-FU perfusion in rabbits [15] have shown endothelial toxicity resulting in disruption of the endothelium of small arteries, patchy exposure of the subendothelium and foci of thrombus formation.
The sinusoidal lesions we observed here were morphologically similar to those seen in veno-occlusive disease, a condition that occurs mainly as a complication of high-dose chemotherapy in the setting of stem-cell transplantation, and recently renamed by DeLeve et al. [1618] as sinusoidal obstruction syndrome (SOS). In our series, the prevalence of SOS in patients who received preoperative chemotherapy was 51%. This figure is substantially higher than the 1020% reported in patients who underwent bone marrow transplantation for lympho-hematological malignancies [17].
The intensity of the sinusoidal dilatation and congestion was occasionally severe, and included the disruption of the sinusoidal wall integrity. This latter finding was well illustrated by the extravasation of erythrocytes in Disses space and by immunohistochemical and ultrastructural studies, which showed the complete absence of endothelial lining in the dilated areas. HSC, however, remain present in the perisinusoidal space, as demonstrated by the preserved expression of CRBP-1, a new marker of HSC [19]. Extensive collagenization of perisinusoidal space and centrilobular vein was occasionally identified. This fibrogenesis was highlighted by the presence of -SM actin-positive HSC, an activation marker of fibrogenic cells, both around sinusoidal dilated areas and in other areas of the lobule. This observation is in agreement with a previous description of veno-occlusive diseases in humans [20].
Based our observations, we propose the hypothesis that an initial toxic injury to the sinusoidal endothelial cells results in sinusoidal wall disruption; this may be followed by an activation of HSC and the deposition of matrix in the sinusoids. Obstruction was caused by erythrocytes sloughing, but also by blebs, characterized by free fragments of cytoplasmic processes, occasionally containing cellular organelles. Ultrastructural bleb formation has been described in an experimental rat model of hepatic sinusoidal drug toxicity [21].
In addition to sinusoidal lesions, we also observed areas of hepatic regeneration, which reached a pattern of NRH in seven cases. NRH has been reported in one published series of patients who received preoperative 5-FU chemotherapy for metastatic carcinoma of the colon, with a frequency equivalent to that of our study [13]. The pathogenesis of NRH is believed to be related to modifications of intrahepatic blood flow [22, 23]. When symptomatic, patients develop signs of portal hypertension and cholestasis with elevated alkaline phosphatase. Despite the fact that NRH could become clinically evident a long time after chemotherapy, the latter is under investigation in our study population, as well as possible delayed hepatic regeneration after surgical resection.
Different chemotherapy protocols for advanced colorectal carcinoma have been developed over the last decade. To date, oxaliplatin, a novel platinum complex, has proven to be a safe and effective therapy for colorectal cancer in combination with 5-FU [24], and its side-effects have been easy to manage with appropriate awareness from patients and care providers. During clinical trials, the adverse events most often reported were hematological toxicity, gastrointestinal tract toxicity and a neuropathy, none of which has been observed with other therapeutic platinum derivatives [25]. In our series, we found a significant correlation between the use of oxaliplatin and the development of severe sinusoidal injury. Carboplatin, another alkylating agent of the cisplatin family, has been rarely associated with the development of veno-occlusive disease in patients who have undergone stem-cell transplantation [26]. However, to our knowledge this is the first report of a series of patients with significant hepatic adverse effects from oxaliplatin. Several interpretations of these data are possible. First, no targeted liver biopsy studies have been performed in patients with colon cancer who have received chemotherapy that included oxaliplatin. Such studies would be difficult to justify from an ethical viewpoint, and may never be performed. For our study we had access to extensive sampling from the resected livers, which allowed accurate histopathological examination and therefore increased the likelihood of detecting even subtle abnormalities. A second possibility is that oxaliplatin alone may in fact have little or no liver toxicity, but in combination with other chemotherapeutic agents (e.g. 5-FU) a synergic effect may occur. This is unlikely, because most chemotherapy protocols for colon cancer include a combination of different drugs, and serious liver toxicity has not been reported. Another explanation may be related to the time these lesions require to develop and progress. In our series we were able to study the evolution of the lesions in few patients who underwent a second liver resection for metastasis recurrence. The sinusoidal lesions persisted for several months after chemotherapy had been discontinued. The histopathological findings suggest that fibrosis persists, and, in the few cases we were able to follow for an extended period (>49 months), may continue to progress and reach considerable extension. The risk of evolution may be important, particularly for those patients who have to receive multiple chemotherapy cycles to obtain better tumor responses. Finally, the relationship between tumoral response and type of chemotherapy received is under study. Preliminary results did not identify a significant difference in the size, localization and number of metastasis, evaluated by computed tomography scan prior to neo-adjuvant therapy, between the control group and the group receiving chemotherapy. No significant relationship has been observed between these tumor characteristics and development of sinusoidal lesion in the non-tumoral liver.
Our ability to evaluate the clinical relevance of our findings was limited by the retrospective nature of this study. One would suspect, however, that the constellation of the lesions described here may result in an impairment of liver function in some patients; it is also possible that the vascular lesions may limit the regenerative ability of the liver after large hepatectomies.
In the light of these findings, we are currently carrying out a prospective study to determine the laboratory and clinical effects of oxaliplatin-induced liver toxicity during the course of and after preoperative chemotherapy. We are also exploring ways to prevent these toxic effects by using glutathione, a compound that has recently been reported to be effective in the prevention of chemotherapy-associated neuropathy [27]. The use of glutathione in an attempt to prevent liver injury has both biological plausibility and experimental support: in a rat model, profound glutathione depletion was found to play a role in the occurrence sinusoidal hepatic toxicity [2830]. Prevention of sinusoidal lesions was also achieved experimentally in rats by maintaining the level of hepatic nitric oxide (NO) through the administration of O(2)-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), a liver-selective NO-donating prodrug, metabolized by hepatic enzymes to release NO within the liver [16, 31]. The use of such drugs in humans has not yet been reported in veno-occlusive disease, but it is conceivable that in the future they may acquire a role in the prevention of vascular liver toxicity.
In conclusion, we have shown that half of the patients who underwent neo-adjuvant chemotherapy, and at least three-quarters of patients who received oxaliplatin, had specific liver lesions. Sinusoidal injury complicated by fibrosis and veno-occlusive lesions should be included in the list of the adverse side-effects of systemic chemotherapy for colorectal tumors, particularly in connection with the use of oxaliplatin. The clinical consequences of these sinusoidal lesions and venular obstruction also need to be evaluated with respect to the potential impairement of liver regeneration after extended resection.
![]() |
Acknowledgements |
---|
![]() |
FOOTNOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Minagawa M, Makuuchi M, Torzilli G et al. Extension of the frontiers of surgical indications in the treatment of liver metastases from colorectal cancer: long-term results. Ann Surg 2000; 231: 487499.[CrossRef][ISI][Medline]
3. Malafosse R, Penna C, Sa Cunha A, Nordlinger B. Surgical management of hepatic metastases from colorectal malignancies. Ann Oncol 2001; 12: 887894.[Abstract]
4. Giacchetti S, Itzhaki M, Gruia G et al. Long-term survival of patients with unresectable colorectal cancer liver metastases following infusional chemotherapy with 5-fluorouracil, leucovorin, oxaliplatin and surgery. Ann Oncol 1999; 10: 663669.[Abstract]
5. Mathijssen RH, van Alphen RJ, Verweij J et al. Clinical pharmacokinetics and metabolism of irinotecan. Clin Cancer Res 2001; 7: 21822194.
6. Carrato A, Gallego J, Diaz-Rubio E. Oxaliplatin: results in colorectal carcinoma. Crit Rev Oncol Hematol 2002; 44: 2944.[ISI][Medline]
7. Saltz LB, Cox JV, Blanke C et al. Irinotecan plus fluorouracil and leucovorin for metastatic colorectal cancer. Irinotecan Study Group. N Engl J Med 2000; 343: 905914.
8. Douillard JY, Cunningham D, Roth AD et al. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet 2000; 355: 10411047.[CrossRef][ISI][Medline]
9. Souglakos J, Mavroudis D, Kakolyris S et al. Triplet combination with irinotecan plus oxaliplatin plus continuous-infusion fluorouracil and leucovorin as first-line treatment in metastatic colorectal cancer: a multicenter phase II trial. J Clin Oncol 2002; 20: 26512657.
10. Giacchetti S, Perpoint B, Zidani R et al. Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracilleucovorin as first-line treatment of metastatic colorectal cancer. J Clin Oncol 2000; 18: 136147.
11. Schmoll HJ. The role of oxaliplatin in the treatment of advanced metastatic colorectal cancer: prospects and future directions. Semin Oncol 2002; 29: 3439.
12. Bismuth H, Adam R. Reduction of nonresectable liver metastasis from colorectal cancer after oxaliplatin chemotherapy. Semin Oncol 1998; 25: 4046.
13. Washington K, Lane KL, Meyers WC. Nodular regenerative hyperplasia in partial hepatectomy specimens. Am J Surg Pathol 1993; 17: 11511158.[ISI][Medline]
14. Kemeny NE, Ron IG. Hepatic arterial chemotherapy in metastatic colorectal patients. Semin Oncol 1999; 26: 524535.[ISI][Medline]
15. Cwikiel M, Zhang B, Eskilsson J et al. The influence of 5-fluorouracil on the endothelium in small arteries. An electron microscopic study in rabbits. Scanning Microsc 1995; 9: 561576.[ISI][Medline]
16. DeLeve LD, Shulman HM, McDonald GB. Toxic injury to hepatic sinusoids: sinusoidal obstruction syndrome (veno-occlusive disease). Semin Liver Dis 2002; 22: 2742.[CrossRef][ISI][Medline]
17. DeLeve LD, McCuskey RS, Wang X et al. Characterization of a reproducible rat model of hepatic veno-occlusive disease. Hepatology 1999; 29: 17791791.[ISI][Medline]
18. Eberhardt W, Beeg T, Beck KF et al. Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells. Kidney Int 2000; 57: 5969.[CrossRef][ISI][Medline]
19. Uchio K, Tuchweber B, Manabe N et al. Cellular retinol-binding protein-1 expression and modulation during in vivo and in vitro myofibroblastic differentiation of rat hepatic stellate cells and portal fibroblasts. Lab Invest 2002; 82: 619628.[ISI][Medline]
20. Sato Y, Asada Y, Hara S et al. Hepatic stellate cells (Ito cells) in veno-occlusive disease of the liver after allogeneic bone marrow transplantation. Histopathology 1999; 34: 6670.[CrossRef][ISI][Medline]
21. Yeong ML, Wakefield SJ, Ford HC. Hepatocyte membrane injury and bleb formation following low dose comfrey toxicity in rats. Int J Exp Pathol 1993; 74: 211217.[ISI][Medline]
22. Wanless IR. Micronodular transformation (nodular regenerative hyperplasia) of the liver: a report of 64 cases among 2,500 autopsies and a new classification of benign hepatocellular nodules. Hepatology 1990; 11: 787797.[ISI][Medline]
23. Shimamatsu K, Wanless IR. Role of ischemia in causing apoptosis, atrophy, and nodular hyperplasia in human liver. Hepatology 1997; 26: 343350.[CrossRef][ISI][Medline]
24. Misset JL, Bleiberg H, Sutherland W et al. Oxaliplatin clinical activity: a review. Crit Rev Oncol Hematol 2000; 35: 7593.[CrossRef][ISI][Medline]
25. Cassidy J, Misset JL. Oxaliplatin-related side effects: characteristics and management. Semin Oncol 2002; 29: 1120.
26. Bearman SI. The syndrome of hepatic veno-occlusive disease after marrow transplantation. Blood 1995; 85: 30053020.
27. Cascinu S, Catalano V, Cordella L et al. Neuroprotective effect of reduced glutathione on oxaliplatin-based chemotherapy in advanced colorectal cancer: a randomized, double-blind, placebo-controlled trial. J Clin Oncol 2002; 20: 34783483.
28. DeLeve LD, Wang X, Kuhlenkamp JF, Kaplowitz N. Toxicity of azathioprine and monocrotaline in murine sinusoidal endothelial cells and hepatocytes: the role of glutathione and relevance to hepatic venoocclusive disease. Hepatology 1996; 23: 589599.[ISI][Medline]
29. DeLeve LD, Wang X. Role of oxidative stress and glutathione in busulfan toxicity in cultured murine hepatocytes. Pharmacology 2000; 60: 143154.[CrossRef][ISI][Medline]
30. Wang X, Kanel GC, DeLeve LD. Support of sinusoidal endothelial cell glutathione prevents hepatic veno-occlusive disease in the rat. Hepatology 2000; 31: 428434.[ISI][Medline]
31. Liu J, Li C, Waalkes MP et al. The nitric oxide donor, V-PYRRO/NO, protects against acetaminophen-induced hepatotoxicity in mice. Hepatology 2003; 37: 324333.[CrossRef][ISI][Medline]