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Effect of single chemotherapeutic agents on the growing skeleton of the rat
Annals of Oncology 11: 1121-1126, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
Original article Effect of single chemotherapeutic agents on the growing skeleton of the rat B. L. van Leeuwen,1 W. A. Kamps,2 R. M. Hartel,1 R. P. H.Veth,3 W. J. Sluiter4 & H. J. Hoekstra1 'Department of Surgical Oncology, 2 Children's Cancer Center, Groningen University Hospital, Groningen; 3Department ofOrthopedics, Nijmegen University Hospital, St Radboud, Nijmegen; ^Internal Medicine, Groningen University Hospital, Groningen, The Netherlands
decreased in the diet controlled groups (P < 0.05). Doxorubicin reduced length growth with 4.12 mm or 18 % (P < 0.05). Background: To establish the effect of chemotherapeutics on Methotrexate reduced length growth with 1.11 mm or 5 % (P < 0.05). Length growth in the cisplatin treated rats did not the growing skeleton, male Wistar rats were studied. Design: Between the ages of 4 and 13 weeks the rats were differ from the diet controls. given i.v. doxorubicin 15 mg/m2 body surface area (BSA), Conclusions: Doxorubicin and methotrexate decrease length methotrexate 60 mg/m2 BSA or cisplatin 7.5 mg/m2 BSA. For growth in the rat tibia by, respectively, 18% and 5%. Cisplatin each group of drug-treated rats there was a diet-matched does not affect length growth. The decrease in growth might be control group that was injected with a placebo only. Rats fed a direct effect of doxorubicin and methotrexate on the tibial ad libitum served as the basic control group for length and growth plate and metaphysis, but may be more pronounced weight growth. Body weight and tibial length were measured due to the malnutrition. weekly. Kidney and liver weight were determined at the end of the study. Results: Weight gain and length growth were significantly Key words: cisplatin, doxorubicin, growth, methotrexate Summary
Introduction In recent years the incidence of childhood cancer has increased [1]. An estimated 1 in 900 people aged 16 to 34 years are survivors of childhood cancer today [2]. As the number of survivors increases, so does knowledge on the adverse side effects of the different treatment modalities. Physicians will change their treatment schedules in order to keep the unwanted side effects to a minimum without compromising the curative intention of the treatment. The deleterious sequelae of cranial radiotherapy have prompted the discontinuation of this treatment modality as a standard for acute lymphoblastic leukemia (ALL), it is currently only used in high risk patients. For pediatric solid tumors more and more emphasis is being put on surgery and chemotherapy as primary (combined) treatment modalities. A lot of attention has been paid to the adverse sequelae of chemotherapy such as infertility, cardiac toxicity and the development of secondary neoplasms. The effect on the skeleton has been somewhat neglected. It is expected that new chemotherapy schedules will be developed in thefightagainst childhood cancer. Therefore it is important to know the effects of different chemotherapeutic agents on the growing skeleton. The aim of this study was to establish the effect of three frequently used chemotherapeutic agents in childhood cancer, doxorubicin (DOX), methotrexate (MTX) and cisplatin (CDDP), on the growing skeleton in the rat.
Materials and methods Rats The study was approved by the Animal Experiments Committee of the Faculty of Medical Sciences of the State University Groningen. Male weanling Wistar albino rats were used. The rats were housed in wool bedded plastic cages, each cage housing six rats. The cages were cleaned twice a week, the light-dark cycle was 10.5-13.5 hours. Temperature and humidity were kept constant at 21 ± 1 °C and 50%. The rats received water at libitum by the water bottle/sipper tube method. Water was acidified to control microbial contaminants. A standard laboratory animal diet was used containing 0.8% calcium and 0.6% phosphorus (food pellet RMH-B, Hope Farms BV, Woerden, the Netherlands). Food hoppers were used to feed the rats and weighed twice weekly. Rats who died or were debilitated to the extent that no normal mobility and food consumption could be expected, were excluded.
Chemotherapeutic agents The chemotherapeutic agents used in the study were: doxorubicin (Adriblastine® provided by Farmitaha Carlo Erba, Rotterdam, the Netherlands), methotrexate (Ledertrexate® provided by Lederle Nederland BV, Etten-Leur, The Netherlands) and cisplatin (Platinol* provided by Bristol-Meyers BV, Weesp, the Netherlands). The body surface area (BSA, m 2 ) was calculated for each rat using the formula: BSA = k. w 2/3 /10 4 , with k = constant (rn^gr 2 ' 3 ) chosen to be 9.5 [3] and w = weight (g). Doses were calculated this way because it allows a relatively good extrapolation of data to humans [4]. Doses selected for the different groups were: 15 mg/m 2 BSA doxorubicin, 60 mg/m 2 BSA methotrexate and 7.5 mg/m 2 BSA cisplatin (Figure 1). These doses were the maximum tolerated dose selected from a pilot study [B. van Leeuwen et al. submitted]. The dose of MTX is compa-
1122 •
Doxorubicin 15 mg/m2 BSA
•
•
•
*
•
•
|....|....|....|....|....|—1....|....|—| 4 6 8 10 12
Table 1. Body weight increase and tibial length growth after administration of doxorubicin, methotrexate or cisplatin to the growing rat. Group
Ad lib
DOX
Diet DOX
MTX
Diet MTX
CDDP
Diet CDDP
n
39
28
28
23
24
33
35
Total weight increase(g)
251 54 134 88" 196.48 203.15 212.18 217.31" 199.58 (26.35) (24.06) (25 57) (33.43) (23 52) (21 31) (18.82)
n
36
—> age (weeks) Methotrexate 60 mg/m2 BSA
• 4
Cisplatin 7.5 mg/m2 BSA
•
•
6
• 8
* •
•
10
12 —> age (weeks) • • • • • • • • • I—|—1___.|—1....|—1._._|....|....| 4 6 8 10 12 —> age (weeks)
Figure 1. Dosing schedules for the different chemotherapeutic agents. * Indicates injection of chemotherapeutic agent or placebo. rable to that used in clinical practice, the other doses are somewhat lower [5] The drugs were administered via puncture of the tail vein. The needle was flushed with 0.5 ml 0.9% NaCl before and after each bolus. Study design A total of 28 rats were treated with doxorubicin, 36 rats received methotrexate and another 36 rats were treated with cisplatin. For each study group there was a diet-controlled group (diet DOX, diet MTX, diet CDDP) of 28, 24 and 36 rats, respectively. The same amount of food that was eaten by the rats receiving a chemotherapeutic agent was given to the rats in the diet-control group. The control group underwent the same procedures as the study group and received equal quantities of a placebo intravenously. The rats were anesthetized weekly with ether, both tibial bones were clipped in an adjustable holder to allow optimal horizontal positioning of the leg. The tibial bones were radiographed with Oralux (Philips, 50 kV, variable exposure time). The distance focus-film was 40 cm,filmsused were Kodak Ultra speed DF45, size 57-76 cm. Tibial length was the distance between the top of the convex curve of the tibial plateau and top of the concavity formed by the distal end of the tibia. The mean value of three measurements on a digitizer was assumed to be sufficient. All rats were weighed weekly. At the start of the experiment all rats were four to five weeks old. One group of forty rats served as a control group to assess basic growth curves for this species under these circumstances. The occurrence of diarrhea was recorded. In all groups survivors were sacrificed at the end of the study aged 13—14 weeks, the liver and kidneys were weighed Calculations and statistical analysis As a /-test showed the left and right tibial lengths were not significantly different (P > 0.05), they were summed This total length was used in further analysis to decrease the error of measurement. The weights of the left and right kidney were also summed and used as kidney weight. For comparison of the different groups in terms of weight gain, length growth and organ weight, /-tests were used. A P-value <0.05 was denominated as statistically significant. To distinguish weight increase due to increase in length from weight increase due to soft tissue or fluid accumulation, the body mass index (BMI) was calculated for all groups using a simplified formula BMI = body weight/total tibia length2.
Results All data used in statistical analysis were normally distributed. At the end of the experiment the rats treated with a chemotherapeutic agent and their diet-matched
Total tibial length growth (mm)
28 21 (196)
28 18.94" (121)
27 23.06 (2.03)
23 s
2138 (2.03)
22
33
35
22.49 (0.84)
21.83 (1.25)
21.84 (1.24)
Results are means, standard deviation in brackets. Abbreviations: ad lib - control rats fed ad libitum; DOX - rats injected with doxorubicin; MTX - rats injected with methotrexate; CDDP - rats injected with cisplatin; diet - diet matched controls for each drug group. a Different from diet matched control group (P < 0.05). All results in study groups differ significantly from the ad lib group (P = 0.000).
controls had gained significantly less weight than the rats fed ad libitum. The total tibial length growth was also significantly less in these rats compared with the rats fed ad libitum (Table 1). Cumulative tibial growth in the different groups is shown in Figure 2. Doxorubicin In the doxorubicin-treated group two rats had diarrhea after the first dose, after the second dose four more rats had diarrhea. After the third and fourth dose no diarrhea was noted, afterfivedoses two rats had diarrhea. To establish the effect of doxorubicin on total tibial length growth, the doxorubicin-treated rats were compared with their diet controls. Total tibial length growth and total weight gain were 4.12 mm (18%) and 61.60 grams (31%) less in the drug treated rats (P - 0.000). Methotrexate After the first injection of methotrexate three rats showed signs of diarrhea, and three more died. During the rest of the experiment no more cases of diarrhea were noted, eight more rats died due to toxicity, one rat died following an overdose of ether. The increase in total tibial length was 1.11 mm (5%) less in the drug treated rats compared to the diet controls (P = 0.022). Weight gain in the diet control group was slightly more than in the drug-treated group (4%) but this difference was not statistically significant (P > 0.05). Cisplatin In the cisplatin-treated rats there were no signs of general toxicity or diarrhea during the experiment. At the end of the experiment the total tibial length growth was not different from the diet-control group and the rats treated with cisplatin had gained 17.73 grams (9%) more in body weight (P - 0.001). This weight increase in the drug-treated group is due to fluid retention as can be seen when comparing the cumulative increase in body
1123 change in BMI cisp latin
doxorubicin
(a) 30 25
1.5
Mo 0.5 8
10 age in weeks doxorubicin
ad libitum
12 •
14
8
diet control
12
14
age In weeks
methotrexate
(b)
10
30
cisplatin
diet (
Figure 3. The cumulative increase in body mass index (BMI) for male Wistar rats receiving cisplatin and their diet matched controls. BMI was calculated using the formula bodywcight in grams/(length left + right tibia in mm) 2 . Dark boxes represent cisplatin treated rats, light boxes are the diet matched controls.
25 E
I 20 §15
f 10
Table 2. Organ weights at 13 weeks of age after administration of doxorubicin, methotrexate or cisplatin to the growing rat.
5 0 8 ad libitum
10
12
age in weeks methotrexate
•
Group
Ad lib
DOX
Diet DOX
MTX
Diet MTX
CDDP
Diet CDDP
n
12
27
27
23
24
33
26
Liver weight (g)
13.68 (1.67)
13.48" (197)
10.99" 11.66" (2 16) (2 48)
11.50" (2 22)
13.53 (144)
13.45 (164)
Kidney weight (g)
2.57 (0.23)
2.67° (0.40)
2.10" (0.23)
2 14" (0 26)
2 13 a - b (0.22)
2.34" (0 23)
14
diet control
cisplatin
125 | 20 |15
2 18" (0 25)
Results are means, standard deviation in brackets. Abbreviations: ad lib - control rats fed ad libitum; DOX - rats injected with doxorubicin, MTX - rats injected with methotrexate, CDDP - rats injected with cisplatin; diet - diet matched controls for each drug group * Different from diet matched control group (P < 0.05). " Different from ad lib group (P < 0.05)
j 10 I 5 0 6 •
8 ad libitum
10 age in weeks cisplatin
12 •
14
diet control
Figure 2. The cumulative length growth of the tibia of male Wistar rats fed ad libitum (ad lib) and of rats receiving doxorubicin, methotrexate or cisplatin injections and their respective diet-control groups. Growth of left and right tibia were summed. The group fed ad libitum did not receive any injections. In every figure the top line represents the group fed at libitum, the middle line represents the diet control group and the bottom line represents the drug treated group. In the bottom figure the line representing growth in the cisplatin treated group is exactly the same for the drug treated group as for the diet control group.
mass index of the treated rats with that of their dietmatched controls (Figure 3). Organ weight Liver weight was significantly decreased in the diet DOX and diet MTX group compared with the ad lib group (P < 0.05) (Table 2). Kidney weight was significantly smaller in all diet-control groups when compared with the ad libitum group (P < 0.05). Compared with the diet controls the kidneys and liver were significantly heavier in the rats treated with doxorubicin (P < 0.05). In the
methotrexate group liver and kidney weight were not influenced by the administration of the chemotherapeutic agent. Rats treated with cisplatin had smaller kidneys than did their diet-matched controls (P < 0.05), liver weight was comparable to that of the diet-control group. Discussion During the time the rats were studied in this investigation they showed rapid growth. The most active growth plates at the distal end of the femur and proximal end of the tibia, stay open throughout life and in this respect they are different from the human growth plate [6, 7]. We are aware of the fact that microknemometry currently seems to be the method most often used to measure limb length in rats. But since we have no experience with this method, we chose one more familiar to us. Rats receiving chemotherapy and their diet-matched controls all grew significantly less in weight and height than the control group fed ad libitum. Rats receiving chemotherapy do not feel well and therefore eat less. In
1124 the 1940s Saxton [8] already showed that tibial growth in rats was suppressed due to a restricted diet by 18%. In this study a restricted diet alone caused a decrease in tibial growth of 18%—23%. Starvation causes the pars anterior of the pituitary gland to atrophy and the release of growth hormone (GH) to be reduced [9]. Circulating levels of insulin-like growth factor (IGF)-l are diminished because of hepatic resistance to GH [10]. The rats receiving MTX or doxorubicin in this study had an additional decrease in tibial length that cannot be explained by reduced food intake alone, but must be a side effect of the chemotherapeutic agent itself. Cases of diarrhea were scarce and it is unlikely that malabsorption was a contributing factor to the growth retardation in the drug treated rats. Several clinical studies have shown the effect of different chemotherapy combinations on growth. GrootLoonen [11] found 6-mercaptopurine and MTX to cause suppression of growth in children with ALL. Halton [12, 13] found chemotherapeutic treatments for ALL including MTX to cause a decrease in bone growth especially during the most intensive year of treatment. This is in accordance with Caruso-Nicoletti [14] who found that during the first year of chemotherapy with prednisone, vincristine, L-asparaginase, daunomicin, MTX and 6-mercaptopurine, growth deceleration occurred. Thereafter catch up growth occurred so that final length in children treated for ALL was not decreased. In contrast there have been reports on normal linear growth in children during treatment with prednisone, vincristine, 6-mercaptopurine, MTX and cyclophosphamide [1517]. Berglund [18] even reported an increase in growth rate during treatment with vincristine, MTX, prednisone and doxorubicin. He believed growth rate was already decreased prior to treatment, due to ALL itself, and that as the disease was being cured, growth rate improved. Recently it was reported that combination chemotherapy hardly influenced growth in children with brain tumors. But the authors acknowledged themselves that the numbers in their study were too small to make any definite statements on this issue [19]. In clinical studies chemotherapeutic schemes consisting of multiple agents are studied. It is extremely difficult to extract the effect of one single chemotherapeutic agent on skeletal growth from the results of such studies. Laboratory studies enable us to study the effect of different antineoplastic drugs in controlled circumstances. Most laboratory studies concerning chemotherapeutic agents have been performed on chondrocytes and osteoblasts in cell cultures or with direct infusion of the drug into the growth plate. Although this gives a clear picture of the effect of chemotherapeutic agents on the growth plate and metaphysis it does not mimic clinical circumstances. The toxic effect of doxorubicin on bone and especially on the osteoblast, causes a decrease in bone mineral density in laboratory animals [20-22]. It is the cause of an increase in the number of pathological fractures seen in patients after administration of this drug [23]. Rainsford [24] reported the inhibition of proteoglycan
resorption and apoptosis of hypertrophic chondrocytes by doxorubicin. Length growth is the result of chondrocytes in the growth plate proliferating and increasing in size. The hypertrophic chondrocytes undergo apoptosis and in the metaphysis bone is formed by osteoblasts. An inhibited apoptosis of chondrocytes results in slower bone formation and this may very well be the explanation for the reduced length growth found in this study. Serum levels of growth hormone, IGF-1 and markers of bone metabolism such as osteocalcin and calcium were not determined in this study, leaving room for further investigations of the underlying mechanism of growth retardation found in different groups. Methotrexate is known to cause osteopathy in children. Osteopathy consists of osteopenia, ricket-like changes in radiographies and bone pain in weight-bearing extremities due to insufficiency and stress fractures [25-27]. MTX also has a toxic effect on osteoblasts causing a decrease in bone mineral density [20, 28, 29]. Its effect is even more pronounced than that of doxorubicin. The number of osteoblasts and osteoclasts is not reduced by MTX [30]. In earlier studies it has been reported that the metabolic rate of bone formation during MTX treatment remains stable, suggesting normal ongoing growth [31, 32]. This is in accordance with Robson [33] who showed that the proliferative response of growth plate chondrocytes is not affected by MTX. Chondrocytes were studied in a suspension of MTX at concentrations of up to 50 ug/ml, comparable to plasma peak levels in vivo. In contrast Moell [34] showed reduced length growth in rabbits after a single dose of 1000 mg/kg MTX. This study showed that although growth persists during MTX treatment, the final length is decreased. The effect is less pronounced than in rats treated with doxorubicin but nevertheless significant. Cisplatin is well known for its nephrotoxic effect, the accompanying hypomagnesemia affects the skeleton. It causes cessation of bone growth with a decrease in activity of osteoblasts and osteoclasts, decreased bone formation and osteopenia [35, 36]. Cisplatin has a cytotoxic effect on growth plate chondrocytes in cell cultures [37]. One would expect with all these results to see some influence of cisplatin on length growth. The absence of such an effect in this study can be possibly explained by the relatively low dose cisplatin. As with the rats in this study, children receiving chemotherapy are often malnourished. Malnourished rats have smaller livers with a decreased dihydroflolate reductase content. This causes decreased excretion of methotrexate into bile and increased plasma concentrations and toxicity of the drug [38-41]. Doxorubicin is inactivated by glycosidase, an enzyme found mainly in the liver and kidney [42]. Compared with the diet-matched controls liver weight in the doxorubicin-treated rats was increased, this may well be due to parenchymal degeneration and swelling [43]. Decreased liver function may increase the serum levels of doxorubicin and the effect of this drug on bone. It is possible that the additional effects caused by malnutrition are the reason we were
1125 able to show an effect on length growth of methotrexate and doxorubicin in this study. In rats treated with cisplatin there was a periodic increase in weight after each dose. Cisplatin is nephrotoxic and the weight increase seen in these rats is due to fluid they accumulate after injection of the drug (Figure 3). Kidney weight in the cisplatin group was reduced compared with that in the diet-matched controls, a sign of nephrotoxicity of cisplatin. In this experimental rat model, doxorubicin and methotrexate decrease length growth in the rat tibia by, respectively, 18% and 5%. In contrast cisplatin does not affect length growth at all. The decrease in growth of the skeleton might be a direct effect of doxorubicin and MTX on the growth plate and metaphysis of the long bones, but may be more pronounced due to the malnutrition. Therefore it seems advisable to improve the nutritional status of children with cancer to decrease these possible side effects of chemotherapeutic agents on the growing skeleton.
Acknowledgement Financial support for this study was provided by the Groningen Foundation for Pediatric Oncology Research (SKOG).
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Original article Effect of single chemotherapeutic agents on the growing skeleton of the rat B. L. van Leeuwen,1 W. A. Kamps,2 R. M. Hartel,1 R. P. H.Veth,3 W. J. Sluiter4 & H. J. Hoekstra1 'Department of Surgical Oncology, 2 Children's Cancer Center, Groningen University Hospital, Groningen; 3Department ofOrthopedics, Nijmegen University Hospital, St Radboud, Nijmegen; ^Internal Medicine, Groningen University Hospital, Groningen, The Netherlands
decreased in the diet controlled groups (P < 0.05). Doxorubicin reduced length growth with 4.12 mm or 18 % (P < 0.05). Background: To establish the effect of chemotherapeutics on Methotrexate reduced length growth with 1.11 mm or 5 % (P < 0.05). Length growth in the cisplatin treated rats did not the growing skeleton, male Wistar rats were studied. Design: Between the ages of 4 and 13 weeks the rats were differ from the diet controls. given i.v. doxorubicin 15 mg/m2 body surface area (BSA), Conclusions: Doxorubicin and methotrexate decrease length methotrexate 60 mg/m2 BSA or cisplatin 7.5 mg/m2 BSA. For growth in the rat tibia by, respectively, 18% and 5%. Cisplatin each group of drug-treated rats there was a diet-matched does not affect length growth. The decrease in growth might be control group that was injected with a placebo only. Rats fed a direct effect of doxorubicin and methotrexate on the tibial ad libitum served as the basic control group for length and growth plate and metaphysis, but may be more pronounced weight growth. Body weight and tibial length were measured due to the malnutrition. weekly. Kidney and liver weight were determined at the end of the study. Results: Weight gain and length growth were significantly Key words: cisplatin, doxorubicin, growth, methotrexate Summary
Introduction In recent years the incidence of childhood cancer has increased [1]. An estimated 1 in 900 people aged 16 to 34 years are survivors of childhood cancer today [2]. As the number of survivors increases, so does knowledge on the adverse side effects of the different treatment modalities. Physicians will change their treatment schedules in order to keep the unwanted side effects to a minimum without compromising the curative intention of the treatment. The deleterious sequelae of cranial radiotherapy have prompted the discontinuation of this treatment modality as a standard for acute lymphoblastic leukemia (ALL), it is currently only used in high risk patients. For pediatric solid tumors more and more emphasis is being put on surgery and chemotherapy as primary (combined) treatment modalities. A lot of attention has been paid to the adverse sequelae of chemotherapy such as infertility, cardiac toxicity and the development of secondary neoplasms. The effect on the skeleton has been somewhat neglected. It is expected that new chemotherapy schedules will be developed in thefightagainst childhood cancer. Therefore it is important to know the effects of different chemotherapeutic agents on the growing skeleton. The aim of this study was to establish the effect of three frequently used chemotherapeutic agents in childhood cancer, doxorubicin (DOX), methotrexate (MTX) and cisplatin (CDDP), on the growing skeleton in the rat.
Materials and methods Rats The study was approved by the Animal Experiments Committee of the Faculty of Medical Sciences of the State University Groningen. Male weanling Wistar albino rats were used. The rats were housed in wool bedded plastic cages, each cage housing six rats. The cages were cleaned twice a week, the light-dark cycle was 10.5-13.5 hours. Temperature and humidity were kept constant at 21 ± 1 °C and 50%. The rats received water at libitum by the water bottle/sipper tube method. Water was acidified to control microbial contaminants. A standard laboratory animal diet was used containing 0.8% calcium and 0.6% phosphorus (food pellet RMH-B, Hope Farms BV, Woerden, the Netherlands). Food hoppers were used to feed the rats and weighed twice weekly. Rats who died or were debilitated to the extent that no normal mobility and food consumption could be expected, were excluded.
Chemotherapeutic agents The chemotherapeutic agents used in the study were: doxorubicin (Adriblastine® provided by Farmitaha Carlo Erba, Rotterdam, the Netherlands), methotrexate (Ledertrexate® provided by Lederle Nederland BV, Etten-Leur, The Netherlands) and cisplatin (Platinol* provided by Bristol-Meyers BV, Weesp, the Netherlands). The body surface area (BSA, m 2 ) was calculated for each rat using the formula: BSA = k. w 2/3 /10 4 , with k = constant (rn^gr 2 ' 3 ) chosen to be 9.5 [3] and w = weight (g). Doses were calculated this way because it allows a relatively good extrapolation of data to humans [4]. Doses selected for the different groups were: 15 mg/m 2 BSA doxorubicin, 60 mg/m 2 BSA methotrexate and 7.5 mg/m 2 BSA cisplatin (Figure 1). These doses were the maximum tolerated dose selected from a pilot study [B. van Leeuwen et al. submitted]. The dose of MTX is compa-
1122 •
Doxorubicin 15 mg/m2 BSA
•
•
•
*
•
•
|....|....|....|....|....|—1....|....|—| 4 6 8 10 12
Table 1. Body weight increase and tibial length growth after administration of doxorubicin, methotrexate or cisplatin to the growing rat. Group
Ad lib
DOX
Diet DOX
MTX
Diet MTX
CDDP
Diet CDDP
n
39
28
28
23
24
33
35
Total weight increase(g)
251 54 134 88" 196.48 203.15 212.18 217.31" 199.58 (26.35) (24.06) (25 57) (33.43) (23 52) (21 31) (18.82)
n
36
—> age (weeks) Methotrexate 60 mg/m2 BSA
• 4
Cisplatin 7.5 mg/m2 BSA
•
•
6
• 8
* •
•
10
12 —> age (weeks) • • • • • • • • • I—|—1___.|—1....|—1._._|....|....| 4 6 8 10 12 —> age (weeks)
Figure 1. Dosing schedules for the different chemotherapeutic agents. * Indicates injection of chemotherapeutic agent or placebo. rable to that used in clinical practice, the other doses are somewhat lower [5] The drugs were administered via puncture of the tail vein. The needle was flushed with 0.5 ml 0.9% NaCl before and after each bolus. Study design A total of 28 rats were treated with doxorubicin, 36 rats received methotrexate and another 36 rats were treated with cisplatin. For each study group there was a diet-controlled group (diet DOX, diet MTX, diet CDDP) of 28, 24 and 36 rats, respectively. The same amount of food that was eaten by the rats receiving a chemotherapeutic agent was given to the rats in the diet-control group. The control group underwent the same procedures as the study group and received equal quantities of a placebo intravenously. The rats were anesthetized weekly with ether, both tibial bones were clipped in an adjustable holder to allow optimal horizontal positioning of the leg. The tibial bones were radiographed with Oralux (Philips, 50 kV, variable exposure time). The distance focus-film was 40 cm,filmsused were Kodak Ultra speed DF45, size 57-76 cm. Tibial length was the distance between the top of the convex curve of the tibial plateau and top of the concavity formed by the distal end of the tibia. The mean value of three measurements on a digitizer was assumed to be sufficient. All rats were weighed weekly. At the start of the experiment all rats were four to five weeks old. One group of forty rats served as a control group to assess basic growth curves for this species under these circumstances. The occurrence of diarrhea was recorded. In all groups survivors were sacrificed at the end of the study aged 13—14 weeks, the liver and kidneys were weighed Calculations and statistical analysis As a /-test showed the left and right tibial lengths were not significantly different (P > 0.05), they were summed This total length was used in further analysis to decrease the error of measurement. The weights of the left and right kidney were also summed and used as kidney weight. For comparison of the different groups in terms of weight gain, length growth and organ weight, /-tests were used. A P-value <0.05 was denominated as statistically significant. To distinguish weight increase due to increase in length from weight increase due to soft tissue or fluid accumulation, the body mass index (BMI) was calculated for all groups using a simplified formula BMI = body weight/total tibia length2.
Results All data used in statistical analysis were normally distributed. At the end of the experiment the rats treated with a chemotherapeutic agent and their diet-matched
Total tibial length growth (mm)
28 21 (196)
28 18.94" (121)
27 23.06 (2.03)
23 s
2138 (2.03)
22
33
35
22.49 (0.84)
21.83 (1.25)
21.84 (1.24)
Results are means, standard deviation in brackets. Abbreviations: ad lib - control rats fed ad libitum; DOX - rats injected with doxorubicin; MTX - rats injected with methotrexate; CDDP - rats injected with cisplatin; diet - diet matched controls for each drug group. a Different from diet matched control group (P < 0.05). All results in study groups differ significantly from the ad lib group (P = 0.000).
controls had gained significantly less weight than the rats fed ad libitum. The total tibial length growth was also significantly less in these rats compared with the rats fed ad libitum (Table 1). Cumulative tibial growth in the different groups is shown in Figure 2. Doxorubicin In the doxorubicin-treated group two rats had diarrhea after the first dose, after the second dose four more rats had diarrhea. After the third and fourth dose no diarrhea was noted, afterfivedoses two rats had diarrhea. To establish the effect of doxorubicin on total tibial length growth, the doxorubicin-treated rats were compared with their diet controls. Total tibial length growth and total weight gain were 4.12 mm (18%) and 61.60 grams (31%) less in the drug treated rats (P - 0.000). Methotrexate After the first injection of methotrexate three rats showed signs of diarrhea, and three more died. During the rest of the experiment no more cases of diarrhea were noted, eight more rats died due to toxicity, one rat died following an overdose of ether. The increase in total tibial length was 1.11 mm (5%) less in the drug treated rats compared to the diet controls (P = 0.022). Weight gain in the diet control group was slightly more than in the drug-treated group (4%) but this difference was not statistically significant (P > 0.05). Cisplatin In the cisplatin-treated rats there were no signs of general toxicity or diarrhea during the experiment. At the end of the experiment the total tibial length growth was not different from the diet-control group and the rats treated with cisplatin had gained 17.73 grams (9%) more in body weight (P - 0.001). This weight increase in the drug-treated group is due to fluid retention as can be seen when comparing the cumulative increase in body
1123 change in BMI cisp latin
doxorubicin
(a) 30 25
1.5
Mo 0.5 8
10 age in weeks doxorubicin
ad libitum
12 •
14
8
diet control
12
14
age In weeks
methotrexate
(b)
10
30
cisplatin
diet (
Figure 3. The cumulative increase in body mass index (BMI) for male Wistar rats receiving cisplatin and their diet matched controls. BMI was calculated using the formula bodywcight in grams/(length left + right tibia in mm) 2 . Dark boxes represent cisplatin treated rats, light boxes are the diet matched controls.
25 E
I 20 §15
f 10
Table 2. Organ weights at 13 weeks of age after administration of doxorubicin, methotrexate or cisplatin to the growing rat.
5 0 8 ad libitum
10
12
age in weeks methotrexate
•
Group
Ad lib
DOX
Diet DOX
MTX
Diet MTX
CDDP
Diet CDDP
n
12
27
27
23
24
33
26
Liver weight (g)
13.68 (1.67)
13.48" (197)
10.99" 11.66" (2 16) (2 48)
11.50" (2 22)
13.53 (144)
13.45 (164)
Kidney weight (g)
2.57 (0.23)
2.67° (0.40)
2.10" (0.23)
2 14" (0 26)
2 13 a - b (0.22)
2.34" (0 23)
14
diet control
cisplatin
125 | 20 |15
2 18" (0 25)
Results are means, standard deviation in brackets. Abbreviations: ad lib - control rats fed ad libitum; DOX - rats injected with doxorubicin, MTX - rats injected with methotrexate, CDDP - rats injected with cisplatin; diet - diet matched controls for each drug group * Different from diet matched control group (P < 0.05). " Different from ad lib group (P < 0.05)
j 10 I 5 0 6 •
8 ad libitum
10 age in weeks cisplatin
12 •
14
diet control
Figure 2. The cumulative length growth of the tibia of male Wistar rats fed ad libitum (ad lib) and of rats receiving doxorubicin, methotrexate or cisplatin injections and their respective diet-control groups. Growth of left and right tibia were summed. The group fed ad libitum did not receive any injections. In every figure the top line represents the group fed at libitum, the middle line represents the diet control group and the bottom line represents the drug treated group. In the bottom figure the line representing growth in the cisplatin treated group is exactly the same for the drug treated group as for the diet control group.
mass index of the treated rats with that of their dietmatched controls (Figure 3). Organ weight Liver weight was significantly decreased in the diet DOX and diet MTX group compared with the ad lib group (P < 0.05) (Table 2). Kidney weight was significantly smaller in all diet-control groups when compared with the ad libitum group (P < 0.05). Compared with the diet controls the kidneys and liver were significantly heavier in the rats treated with doxorubicin (P < 0.05). In the
methotrexate group liver and kidney weight were not influenced by the administration of the chemotherapeutic agent. Rats treated with cisplatin had smaller kidneys than did their diet-matched controls (P < 0.05), liver weight was comparable to that of the diet-control group. Discussion During the time the rats were studied in this investigation they showed rapid growth. The most active growth plates at the distal end of the femur and proximal end of the tibia, stay open throughout life and in this respect they are different from the human growth plate [6, 7]. We are aware of the fact that microknemometry currently seems to be the method most often used to measure limb length in rats. But since we have no experience with this method, we chose one more familiar to us. Rats receiving chemotherapy and their diet-matched controls all grew significantly less in weight and height than the control group fed ad libitum. Rats receiving chemotherapy do not feel well and therefore eat less. In
1124 the 1940s Saxton [8] already showed that tibial growth in rats was suppressed due to a restricted diet by 18%. In this study a restricted diet alone caused a decrease in tibial growth of 18%—23%. Starvation causes the pars anterior of the pituitary gland to atrophy and the release of growth hormone (GH) to be reduced [9]. Circulating levels of insulin-like growth factor (IGF)-l are diminished because of hepatic resistance to GH [10]. The rats receiving MTX or doxorubicin in this study had an additional decrease in tibial length that cannot be explained by reduced food intake alone, but must be a side effect of the chemotherapeutic agent itself. Cases of diarrhea were scarce and it is unlikely that malabsorption was a contributing factor to the growth retardation in the drug treated rats. Several clinical studies have shown the effect of different chemotherapy combinations on growth. GrootLoonen [11] found 6-mercaptopurine and MTX to cause suppression of growth in children with ALL. Halton [12, 13] found chemotherapeutic treatments for ALL including MTX to cause a decrease in bone growth especially during the most intensive year of treatment. This is in accordance with Caruso-Nicoletti [14] who found that during the first year of chemotherapy with prednisone, vincristine, L-asparaginase, daunomicin, MTX and 6-mercaptopurine, growth deceleration occurred. Thereafter catch up growth occurred so that final length in children treated for ALL was not decreased. In contrast there have been reports on normal linear growth in children during treatment with prednisone, vincristine, 6-mercaptopurine, MTX and cyclophosphamide [1517]. Berglund [18] even reported an increase in growth rate during treatment with vincristine, MTX, prednisone and doxorubicin. He believed growth rate was already decreased prior to treatment, due to ALL itself, and that as the disease was being cured, growth rate improved. Recently it was reported that combination chemotherapy hardly influenced growth in children with brain tumors. But the authors acknowledged themselves that the numbers in their study were too small to make any definite statements on this issue [19]. In clinical studies chemotherapeutic schemes consisting of multiple agents are studied. It is extremely difficult to extract the effect of one single chemotherapeutic agent on skeletal growth from the results of such studies. Laboratory studies enable us to study the effect of different antineoplastic drugs in controlled circumstances. Most laboratory studies concerning chemotherapeutic agents have been performed on chondrocytes and osteoblasts in cell cultures or with direct infusion of the drug into the growth plate. Although this gives a clear picture of the effect of chemotherapeutic agents on the growth plate and metaphysis it does not mimic clinical circumstances. The toxic effect of doxorubicin on bone and especially on the osteoblast, causes a decrease in bone mineral density in laboratory animals [20-22]. It is the cause of an increase in the number of pathological fractures seen in patients after administration of this drug [23]. Rainsford [24] reported the inhibition of proteoglycan
resorption and apoptosis of hypertrophic chondrocytes by doxorubicin. Length growth is the result of chondrocytes in the growth plate proliferating and increasing in size. The hypertrophic chondrocytes undergo apoptosis and in the metaphysis bone is formed by osteoblasts. An inhibited apoptosis of chondrocytes results in slower bone formation and this may very well be the explanation for the reduced length growth found in this study. Serum levels of growth hormone, IGF-1 and markers of bone metabolism such as osteocalcin and calcium were not determined in this study, leaving room for further investigations of the underlying mechanism of growth retardation found in different groups. Methotrexate is known to cause osteopathy in children. Osteopathy consists of osteopenia, ricket-like changes in radiographies and bone pain in weight-bearing extremities due to insufficiency and stress fractures [25-27]. MTX also has a toxic effect on osteoblasts causing a decrease in bone mineral density [20, 28, 29]. Its effect is even more pronounced than that of doxorubicin. The number of osteoblasts and osteoclasts is not reduced by MTX [30]. In earlier studies it has been reported that the metabolic rate of bone formation during MTX treatment remains stable, suggesting normal ongoing growth [31, 32]. This is in accordance with Robson [33] who showed that the proliferative response of growth plate chondrocytes is not affected by MTX. Chondrocytes were studied in a suspension of MTX at concentrations of up to 50 ug/ml, comparable to plasma peak levels in vivo. In contrast Moell [34] showed reduced length growth in rabbits after a single dose of 1000 mg/kg MTX. This study showed that although growth persists during MTX treatment, the final length is decreased. The effect is less pronounced than in rats treated with doxorubicin but nevertheless significant. Cisplatin is well known for its nephrotoxic effect, the accompanying hypomagnesemia affects the skeleton. It causes cessation of bone growth with a decrease in activity of osteoblasts and osteoclasts, decreased bone formation and osteopenia [35, 36]. Cisplatin has a cytotoxic effect on growth plate chondrocytes in cell cultures [37]. One would expect with all these results to see some influence of cisplatin on length growth. The absence of such an effect in this study can be possibly explained by the relatively low dose cisplatin. As with the rats in this study, children receiving chemotherapy are often malnourished. Malnourished rats have smaller livers with a decreased dihydroflolate reductase content. This causes decreased excretion of methotrexate into bile and increased plasma concentrations and toxicity of the drug [38-41]. Doxorubicin is inactivated by glycosidase, an enzyme found mainly in the liver and kidney [42]. Compared with the diet-matched controls liver weight in the doxorubicin-treated rats was increased, this may well be due to parenchymal degeneration and swelling [43]. Decreased liver function may increase the serum levels of doxorubicin and the effect of this drug on bone. It is possible that the additional effects caused by malnutrition are the reason we were
1125 able to show an effect on length growth of methotrexate and doxorubicin in this study. In rats treated with cisplatin there was a periodic increase in weight after each dose. Cisplatin is nephrotoxic and the weight increase seen in these rats is due to fluid they accumulate after injection of the drug (Figure 3). Kidney weight in the cisplatin group was reduced compared with that in the diet-matched controls, a sign of nephrotoxicity of cisplatin. In this experimental rat model, doxorubicin and methotrexate decrease length growth in the rat tibia by, respectively, 18% and 5%. In contrast cisplatin does not affect length growth at all. The decrease in growth of the skeleton might be a direct effect of doxorubicin and MTX on the growth plate and metaphysis of the long bones, but may be more pronounced due to the malnutrition. Therefore it seems advisable to improve the nutritional status of children with cancer to decrease these possible side effects of chemotherapeutic agents on the growing skeleton.
Acknowledgement Financial support for this study was provided by the Groningen Foundation for Pediatric Oncology Research (SKOG).
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