Effects of 1,2-Dimethylhydrazine Treatment and Feeding ......Sixty-five male Sprague-Dawley rats 4 to 6 weeks old (mean weight, 112 g; Simonsen, Gilroy, Calif.) were divided randomly

[CANCER RESEARCH 43. 1153-1162, March 1983]0008-5472/83/0043-OOOOS02.00

Effects of 1,2-Dimethylhydrazine Treatment and Feeding Regimen on RatColonie Epithelial Cell Proliferation1

David W. Heitman,1-2 Barry G. Grubbs, Teri O. Heitman, and Ivan L. Cameron

Department of Anatomy, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284

ABSTRACT

The present study was designed to test the hypothesis that1,2-dimethylhydrazine dihydrochloride (DMH) induces preneoplasia in rat colonie epithelium and that this DMH-altered epi

thelium will respond differently to various nutritional challengesin comparison to normal colonie epithelium. Preneoplasia wasarbitrarily defined as an altered and irreversible state of colonieepithelial cell proliferation induced by a carcinogen (DMH). Insummary, DMH was found to be specific for the enhancementof rat colonie epithelial cell proliferation compared to otherrapidly renewing cell populations, i.e., ileal epithelium and earepidermis. DMH-induced changes in rat colonie epithelial cell

proliferation and crypt cellularity were found to be irreversiblefollowing a 2- to 8-week recovery period.

The p.o. administration of the solid and liquid diets, regardless of chemical constituents, supported a DMH-induced increase in colonie epithelial cell proliferation; however, a DMH-

induced increase in epithelial cell proliferation was not observed in rats maintained on total parenteral nutrition. Thus,the route of administration has a significant influence on epithelial cell proliferation in colonie epithelium of DMH-treatedrats.

The importance of these results, along with previous studies,is the establishment and initial characterization of an exploitable preneoplastic system in rat colonie epithelium. Particularlyrevealing was the finding that significant changes in cryptkinetic parameters induced by DMH treatment did not revert tocontrol values following a 2- to 8-week recovery period. Based

on an altered and irreversible state of colonie epithelial cellproliferation induced by DMH, it is concluded that: (a) thepreneoplastic state is a committed state and is not dependentupon the continued presence of the carcinogen; and (b) allcryptai epithelium is preneoplastic, although not all cells progress to the overtly transformed state. In addition, total parenteral nutrition prevented the expression of a DMH-induced

preneoplastic state of altered epithelial cell proliferation.

INTRODUCTION

DMH3 has been shown to induce colonie carcinomas in rats

and mice with marked organ specificity (7, 26, 27, 44, 59, 68,72, 81). A moderate dose of DMH (7 to 14 mg/kg weekly)produces almost exclusively colon tumors (26, 63, 64, 80).The results of previous studies using mice and rats have alsoshown that s.c. DMH injections lead to an increase in colonie

1 Supported by American Cancer Society Institutional Grant IH-116.2 To whom requests for reprints should be addressed, at Department of

Medicine, Division of Gastroenterology, The University of Texas Health Science, Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284.

3The abbreviations used are: DMH, 1,2-dimethylhydrazine dihydrochloride;

TPN. total parenteral nutrition.Received February 12, 1982; accepted October 22, 1982.

crypt cell proliferation, colonie crypt proliferative zone size,and colonie crypt cellularity prior to the development of focalatypias (23, 44, 65, 74).

Although several studies have investigated the influence ofdifferent nutritional factors on the incidence of DMH-inducedcolonie adenocarcinomas, none has investigated the possibleinteraction between DMH treatment and feeding regimen onepithelial cell proliferation in the rat descending colon. Thepresent study is designed to detect any such interaction.

The specific aims of the present study are to investigate: (a)the organ specificity of DMH action on the enhancement of ratcolonie epithelial cell proliferation compared to other rapidlyrenewing cell populations; (b) the influence of 3 different iso-calorie feeding regimens on epithelial cell proliferation andcrypt cellularity in the rat descending colon and distal ileum;(c) the interaction between DMH treatment and feeding regimens on rat colonie epithelial cell proliferation and crypt cellularity; and (d) the reversibility of DMH-induced changes in rat

colonie epithelial cell proliferation and crypt cellularity.

MATERIALS AND METHODS

Animals and Treatments

Sixty-five male Sprague-Dawley rats 4 to 6 weeks old (mean weight,

112 g; Simonsen, Gilroy, Calif.) were divided randomly into 2 groups.One group of rats was given 8 weekly s.c. injections of DMH at adosage of 9.5 mg/kg body weight. Injections were given at weeklyintervals, because it has been shown to be the time needed for thecolonie mucosa to recover following the acute toxic effects of a singleinjection of DMH (48). The DMH (Aldrich Chemical Co., Inc., Milwaukee, Wis.) was dissolved at a concentration of 2.1 g (0.95 g DMH) per100 ml of a vehicle solution containing 0.18% EDTA added as astabilizing agent. The pH was adjusted to 6.5 with NaOH. Control rats(non-DMH) were given injections of the same volume of the vehicle

solution. Animals were given solid food (Wayne Lab Blox; Allied Mills,Inc., Chicago, III.) and water ad libitum and were housed in a roommaintained at 25Â°with 14 hr of light and 10 hr of darkness throughout

the 8-week DMH injection period.

Feeding Regimens

One week after the last injection of DMH, rats with and without DMHtreatments were randomly divided into 12 groups. Six groups wereused in the experimental design to test the reversibility of DMH effectson parameters measured in the epithelium of the rat descending colon.These rats remained on solid food ad libitum for either 2, 4, or 8 weeksafter the last DMH injection. The remaining 6 groups were placed onone of 3 different isocaloric feeding regimens for 10 days. The differentfeeding regimens included: (a) solid food ad libitum (Wayne Lab Blox,3.04 kcal/g); (b) a complete liquid elemental diet given ad libitum(1.24 kcal/ml); and (c) TPN with the same complete elemental diet.The liquid elemental diet used contained 27% glucose, 3.8% crystallineessential and nonessential amino acids (Freamine; McGaw Laboratories, Irvine, Calif.), as well as appropriate electrolytes, vitamins, andtrace elements. Water was available to all rats ad libitum. The rats on

MARCH 1983 1153

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D. W. Heitman et al.

these different feeding regimens were individually housed in metaboliccages throughout the 10-day duration of the feeding regimens in aroom maintained at 25Â°with 14 hr of light and 10 hr of darkness. The

metabolic cages had wide-mesh bottoms to prevent coprophagy.

Caloric intake and body weight were monitored for each rat on thedifferent feeding regimens. Table 1 shows a comparison between meandaily caloric intake as analyzed by one-way analysis of variance. There

were no significant differences in the mean caloric intake between ratson the different feeding regimens. The results of one-way analysis of

variance on rat body weight change, expressed as a ratio of final bodyweight to initial body weight, showed no significant differences amongthe groups of rats listed in Table 1 (mean Â±S.E. for Groups 1 to 6:1.048 Â± 0.006; 1.038 Â± 0.007; 1.043 Â± 0.010; 1.023 Â± 0.005;1.036 Â±0.008; and 1.040 Â±0.012, respectively; F value = 1.17).

Techniques for TPN

The techniques, solutions, and equipment for TPN of unrestrainedrats have been described in detail elsewhere (69) and have been usedsuccessfully in several recent studies to maintain proper growth and apositive nitrogen balance (9-14, 34, 35, 38, 56). The method involves

insertion of a Silastic catheter tube into the superior vena cava or theright atrium via the external jugular vein while the rats were under etheranesthesia. The other end of the catheter is tunneled s.c. and made toexit the body between the scapulae. The rat is placed in a paddedharness and attached to a fluid infusion swivel apparatus (Instec Labs,Philadelphia, Pa.) via a flexible but tightly wound steel shielding cable,which protects the plastic infusion tubing. The i.v. solution is continuously infused with a Holier Model 903 pump. The rats fed P.O. weresham operated and then harnessed before being placed on the soliddiet or the liquid diet.

Tissue Preparation

Three hr prior to killing the rats, each rat received an i.p. injection ofcolchicine at a dosage of 1 mg/kg body weight. The rationale anddetails of this technique, which arrests cells at metaphase and thereforecollects all cells which enter metaphase over a 3-hr period, has been

described by Cameron (8). Animals were killed by an ether overdose.The following tissues were excised for histology: descending colon;distal ileum; and ear. The descending colon was removed, openedlongitudinally, and pinned onto corkboard, serosal side down. For lightmicroscopy, the entire descending colon was fixed in 3% paraformal-dehyde in 0.1 M phosphate buffer (pH 7.3) for 24 hr at 4Â°. The

gastrointestinal tract was examined for any macroscopic lesions. A 5-

mm segment of the distal colon taken 2 cm above the level of the pelvicrim was embedded in paraffin, and 4-/im-thick sections were prepared.

Slides prepared for histological analysis were stained with hematoxylinand eosin.

Segments of the distal ileum 1 cm in length, taken 1 cm above theileocecal junction, were opened longitudinally, laid out serosal sidedown on paper cards, and fixed in 10% formaldehyde in 0.1 M phosphate buffer (pH 7.3). Tissues were embedded in paraffin and sectioned at 4 Â¡on.Sections were stained with hematoxylin and eosin.Pieces 1 cm from the tip of the ear were also fixed and prepared forlight microscopic analysis.

Tissue Analysts

Light Microscopy. For light microscopic analysis of the descendingcolon, 20 complete longitudinal crypt sections were used to obtain amean value for epithelial parameters measured in each rat. A longitudinal crypt section was considered complete if the lumen was exposedfrom the top to the base of the crypt. The sections selected forexamination were spaced so that each crypt section was encounteredonly once. For scoring and analysis, each longitudinal crypt sectionwas divided at the base into 2 crypt columns. Starting at the base ofthe crypt column, nuclei were numbered up to the luminal surface ofthe colon, and the number and position of metaphase figures werescored. Crypt column height was measured in number of cells from thebase to the surface and in mm, using a calibrated ocular micrometer.The height of the proliferative zone was expressed as the highest cellposition of observed metaphase figures. Proliferative zone size wascalculated as the highest cell position of observed metaphase figuresexpressed as the percentage of the crypt column height in number ofcells. The mitotic index was expressed as the number of metaphasefigures in a longitudinal crypt per total number of cells in a longitudinalcrypt section x 100. Additionally, the mitotic index was expressed asthe number of metaphase figures in a longitudinal crypt divided by thehighest cell position of observed metaphase figures x 100. In this way.a mitotic index was obtained for the whole crypt and for the proliferativezone. A total number of metaphase figures scored in each cell positionof 20 complete crypts per rat was obtained, and from these data, amean cell position of metaphase figures measured from the crypt basewas calculated for each group of rats.

The parameters scored in the rat distal ileum were: number ofmetaphase figures per longitudinal crypt section; crypt column heightin number of cells; and crypt column height measured in mm. Themitotic index was expressed as the number of metaphase figures pertotal number of cells in a longitudinal crypt section x 100. Twentycomplete ileal crypts per rat were used in the analysis. The mitoticindex for the ear epidermis was obtained by scoring the number ofmetaphase figures per total number of ear epithelial cells x 100. Thiswas done around the circumference of a cross-section of ear for each

animal.

Statistical Design for Analysis of Data

For statistical analysis of the light microscopic data obtained from

Table 1

Comparison between mean daily caloric intake of different feeding regimens

Rats were given 8 weekly injections of either DMH or vehicle. One week after the last injection, rats wereplaced on one of 3 different isocaloric feeding regimens for 10 days. Values for kcal/day/g body weightwere subjected to one-way analysis of variance.

Group123456Feeding regimenSolid

dietp.o.Soliddietp.o.Liquid

elemental dietp.o.Liquidelemental dietp.o.Liquidelementaldieti.V.â€”

TPNLiquid

elementaldieti.V.â€”TPNTreat

mentVehicleDMHVehicleDMHVehicleDMHkcal/day/gbody

wt0.152Â±0.004a0.156

Â±0.0040.153Â±0.0070.152Â±0.0040.152Â±0.0030.163

Â±0.002No.

ofrats1416121355

One-way analysis of variance (F value)

0.36"

Mean Â±S.E.' Not significant.

1154 CANCER RESEARCH VOL. 43



DMH and Feeding Regimens on Colonie Cell Proliferation

rats on the different feeding regimens (Table 1), 2 independent experimental variables were arranged in a 2 x 3 factorial design. The firstindependent variable, type of treatment, consisted of (a) DMH treatmentand (to) non-DMH treatment. The second independent variable, type of

feeding regimen, consisted of (a) a P.O. solid feeding regimen, (to) aP.O. liquid feeding regimen, and (c) a TPN feeding regimen. Therefore,there were 2 levels of the first independent variable (type of treatment)and 3 levels of the second independent variable (type of feedingregimen). The different parameters measured in the descending colon,the distal ileum, and the ear were referred to as dependent variables inthe statistical design. The dependent variables in the statistical designfor the descending colon data were: (a) mean number of metaphasefigures; (to) mean crypt metaphase index; (c) mean crypt column heightmeasured in mm; (d) mean crypt column height measured in numberof cells; (e) mean proliferative zone height measured as the meanhighest cell position of observed metaphase figures; ( 0 mean proliferative zone size calculated as the mean highest cell position ofobserved metaphase figures expressed as the percentage of the cryptcolumn height in number of cells; (g) mean metaphase index of theproliferative zone; and (h) mean cell position of metaphase figuresmeasured from the crypt base.

The dependent variables in the statistical design for the distal ileumdata were: (a) mean number of metaphase figures; (to) mean cryptmetaphase index; (c) mean crypt column height measured in mm; and(d) mean crypt column height measured in number of cells. Thedependent variable in the statistical design for the ear data was themean metaphase index of the ear epidermis.

The 2-way analysis of variance as described in detail by Huck ef al.

(39) was used to answer 3 research questions. In brief, 2 researchquestions involved the marginal row means and the marginal columnmeans. If a significant difference was found between the marginalmeans for the levels of an independent variable, then it was reportedas a significant main effect of that independent variable acting separately on a dependent variable. A third research question involved cellmeans within the 2 x 3 design. This third question was concerned withthe possible existence of an interaction between the 2 independentvariables. If an interaction occurred in the 2-way analysis of variance,

it was the effect of 2 independent variables operating together on adependent variable.

When there was a significant main effect of feeding regimen, withoutany interaction, a multiple comparison procedure was appropriate fordetermining which feeding regimens were significantly different fromone another across both levels of treatment. If a significant interactionoccurred in the 2-way analysis of variance, then a comparison of simple

main effects was made instead of a comparison of marginal row meansor marginal column means. This involved a comparison of the levels ofone independent variable at each separate level of the other independent variable.

For statistical analysis of the light microscopic data obtained fromrats allowed either 2, 4, or 8 weeks recovery from DMH treatment, 2independent variables were arranged in a 2 x 3 factorial design. Thefirst independent variable, type of treatment, consisted of (a) DMHtreatment and (to) non-DMH treatment. The second independent vari

able, recovery time, consisted of (a) 2 weeks, (to) 4 weeks, and (c) 8weeks. The dependent variables in the statistical design for the descending colon were: (a) mean number of metaphase figures; (to) meancrypt metaphase index; (c) mean crypt column height measured in mm;and (d) mean crypt height measured in number of cells. The samecriteria, as described previously, were used for all follow-up analyses.

BMDP and SPSS statistical programs were used for 2-way analysisof variance and for one-way analysis of variance and Student-Newman-Keul's multiple comparison tests when appropriate (25, 53). A p value

of <0.05 was considered to be significant.

RESULTS

Effects of DMH Treatment and Feeding Regimen

Colonie Epithelium. The results of 2-way analysis of vari

ance of crypt cellularity and epithelial cell proliferation in therat descending colon are summarized in Tables 2 to 4. Therewas a significant main effect of treatment on crypt cellularity asshown in Tables 2 and 3. DMH-treated rats showed a significant

increase in marginal column means for these parameters, ascompared to non-DMH-treated rats, across all feeding regi

mens. Additionally, there was a significant main effect of feeding regimen on all crypt cellularity parameters, except forproliferative zone size. The results of Student-Newman-Keul's

multiple comparison test for feeding regimen marginal rowmeans showed rats on the solid feeding regimen to be significantly higher with respect to crypt cellularity as compared torats maintained on the liquid feeding regimen and rats maintained on the TPN feeding regimen, across both treatments.There were no significant differences between rats on the liquidfeeding regimen and rats on the TPN feeding regimen.

There was a significant main effect of treatment on the

Table 2

Crypt column height in the rat descending colon

Rats were given 8 weekly injections of either DMH or vehicle. One week after the last injection, rats were placed on one of 3 differentisocaloric feeding regimens for 10 days. Rats were killed 3 hr after receiving an i.p. injection of colchicine (1 mg/kg body weight). Values foreach epithelial parameter were subjected to 2-way analysis of variance.

Crypt column ht (mm) Crypt column ht (no. of cells)

TreatmentFeeding

regimenSolid

p.o.Liquid p.o.TPNNon-DMH0.233

Â±0.020a (14)"

0.207 Â±0.026 (12)0.202 Â±0.032 (5)DMH0.255

Â±0.031 (16)0.218 Â±0.025(13)0.216

Â±0.021 (5)Row

mean0.245

0.2120.209TreatmentNon-DMH33.27

Â±1.2530.98 Â±2.0431.98 Â±2.27DMH37.60

Â±3.2033.40 Â±2.0233.63 Â±2.39Row

mean35.58

32.2432.81

Column mean 0.218 0.235 32.17 35.41

2-way analysis of variancestatisticsTreatment

Feeding regimenTreatment x Feeding regimenF4.51

12.650.27P<0.05

<0.001NSCF19.24

14.601.82P<0.001

<0.001NS

Â°Mean Â±S.D.6 Numbers in parentheses, number of rats.0 NS, not significant.

MARCH 1983 1155




colonie epithelial cell proliferation as summarized in Table 4.There was a significant interaction between treatment andfeeding regimen for the number of metaphase figures per cryptand the proliferative zone metaphase index; therefore, a comparison of simple main effects was performed. This involvedcomparing each of the 2 treatments for each separate feedingregimen, using one-way analysis of variance and Student-New-man-Keul's multiple comparison test.

The results of these analyses for the proliferative zone metaphase index are shown in Chart 1. DMH-treated rats on thesolid feeding regimen and the liquid feeding regimen showeda significant increase in the number of metaphase figures andproliferative zone metaphase index, compared to non-DMH-

treated rats on the same feeding regimen. Rats maintained on

the TPN feeding regimen did not show a significant increase ineither parameter due to DMH treatment.

Heal Epithelium and Ear Epidermis. There was no significantmain effect of treatment, although there was a significant maineffect of feeding regimen on crypt cellularity and epithelial cellproliferation in the rat distal ileum, as summarized in Tables 5and 6. Multiple comparison tests for the feeding regimen rowmeans showed rats on the solid feeding regimen to be significantly higher, with respect to epithelial cell proliferation andcrypt cellularity, than the rats on the liquid feeding regimen andrats on the TPN feeding regimen, across both treatments.

There was a significant main effect of treatment but notfeeding regimen on the metaphase index in the rat ear epidermis as shown in Table 7. DMH-treated rats showed a depressed

Table 3

Proliferative zone height, proliferative zone size, and mean cell position of metaphase figures in the rat descending colon

Rats were given 8 weekly injections of either DMH or vehicle. One week after the last injection, rats were placed on one of 3 different isocaloric feeding regimensfor 10 days. Rats were killed 3 hr after receiving an i.p. injection of colchicine (1 mg/kg body weight). Values for each epithelial parameter were subjected to 2-wayanalysis of variance.

Proliferative zonehtaFeeding

regimenSolid

P.O.Liquid p.o.TPNTreatmentNon-DMH14.59

Â±13.20 Â±12.77 Â±2.76C

1.353.27(14)d

18.88

(12) 16.28(5) 15.94DMHÂ±

3.32(16)Â±2.54(13)Â±2.44 (5)Row

mean16.88

14.8014.35Proliferative

zonesize6TreatmentNon-DMH43.79

Â±42.59 Â±39.64 Â±7.76

3.138.33DMH50.05

Â±5.4248.63 Â±6.0847.19 Â±4.24Row

mean47.13

45.7343.41Cell

position of metaphase figures fromcryptbaseTreatmentNon-DMH

DMH10.66

Â±1.49 12.12 Â±1.809.78 Â±1.05 10.87 Â±1.638.93 Â±1.10 10.80 Â±1.11Row

mean11.44

10.359.86

Column mean 13.76 17.45 42.66 49.08 10.04 11.45

2-way analysis of variance statistics

TreatmentFeeding regimenTreatment x

Feeding regimenF22.08

5.000.40P<0.001

<0.01NSF15.75

1.320.06P<0.001

NSe

NSF13.895.580.27P<0.001

<0.01NS

Mean highest cell position of metaphase figures from crypt base.6 Mean highest cell position of metaphase figures expressed as the percentage of crypt column height in number of cells.c Mean Â±S.D." Numbers in parentheses, number of rats.8 NS. not significant.

Table 4

Crypt proliferative parameters in the rat descending colon

Rats were given 8 weekly injections of either DMH or vehicle. One week after the last injection, rats were placed on one of 3 different isocaloric feeding regimensfor 10 days. Rats were killed 3 hr after receiving an i.p. injection of colchicine (1 mg/kg body weight). Values for each epithelial parameter were subjected to 2-wayanalysis of variance.

No. of metaphase figures/crypt Crypt metaphase index Proliferative zone metaphase index

TreatmentFeeding

regimenSolid

p.o.Liquid p.o.TPNNon-DMH3.64

Â±1.1 7a (14)"

3.33 Â±0.85 (12)3.42 Â±1.10 (5)DMH6.78

Â±2.15(16)5.18 Â±1.27(13)4.15 Â±1.17 (5)Row

mean5.31


Â±1.755.38 Â±1.445.28 Â±1.42DMH8.99

Â±2.767.77 Â±1.936.10 Â±1.31Row

Mean7.34


Â±5.3624.93 Â±5.9826.41 Â±3.77DMH36.06

Â±9.9831.72 Â±6.1825.38 _ 3.55Row

Mean30.69

28.4625.90

Column mean 3.48 5.78 5.40 8.10 25.00 32.83

2-way analysis of variance statistics

TreatmentFeeding regimenTreatment x

Feeding regimenF22.42

4.883.01p<0.001<0.01 <0.05F16.28

2.341.81P<0.001

NSCNSF20.371.773.18P<0.001

NS<0.05

" Mean Â±S.D.0 Numbers in parentheses, number of rats.c NS, not significant.




ear epidermal metaphase index, compared to non-DMH rats

across all 3 feeding regimens.

Responses of Colonie Epithelium after 2, 4, or 8 Weeks ofRecovery from DMH Treatment

The results of 2-way analysis of variance of crypt cellularity

and epithelial cell proliferation in the descending colon fromrats allowed either a 2-, 4-, or 8-week recovery period after the

last DMH injection are listed in Tables 8 and 9. There was asignificant main effect of treatment on all parameters, althoughthere was no significant main effect of recovery time. Therewas no significant interaction between treatment and recoverytime for any of the dependent variables measured in the descending colon. DMH treatment caused a significant increasein both epithelial cell proliferation and crypt cellularity compared to non-DMH treatment across all recovery periods.

DISCUSSION

DMH Specificity of Action for Rat Colonie Epithelium. Astrong case for DMH specificity of action on rat (27, 45, 46,57, 59, 68, 74-77, 84) and mouse (16, 22, 29, 36, 72, 80,

81) colonie epithelium can be made from a review of theliterature and the present findings.

The results of past studies indicate that factors other thanthe extent or type of DMA alkylations must have an influenceon the high incidence of colonie tumors obtained after DMHtreatment (58, 67). Delayed or incomplete repair of damagedDMA in the colon compared to other organs may be an explanation for the high incidence of DMH-induced colonie tumors.

Delayed repair of colonie DNA compared to small intestinalDMA has been observed in rats after a single injection of DMH(42). Unrepaired DNA damage in colonie epithelial cells couldpossibly enhance tumor formation in the colon compared to thesmall intestine.

One explanation for the differential susceptibility to DMHaction between the colon and the small intestine is that theintestinal flora (3, 43, 62) and the bile acids (3,18, 51, 54) caninfluence the incidence of colonie tumors. However, previousstudies (3, 43, 62) have shown that colonie tumors can be


induced in germ-free rats and in segments of rat intestine distal

to a colostomy (15, 83). Gennaro et al. (33) demonstrated that,following treatment of rats with azoxymethane, tumors are

50-,

40-

30-

20-

IO-

p<o.ooi

h

1

p<o.ooir"

II NS

r ixt

n= 14 16

Oral

Solid

12 13Oral

Liquid

Feeding Regimen

5 5

TPN

Chart 1. Comparison of proliferate zone metaphase indices between DMH-treated and non-DMH-treated rats on one of 3 different isocaloric feedingregimens. Rats were given 8 weekly injections of either DMH or the vehicle. Oneweek after the last injection, rats were placed on one of 3 different isocaloricfeeding regimens for 10 days. Rats were killed 3 hr after receiving an Â¡.p.injectionof colchicine (1 mg/kg body weight). The proliferate zone metaphase indexwas calculated as the number of metaphasefigures in a longitudinal crypt sectionexpressed as the percentage of the highest cell position of observed metaphasefigures. Columns, means; oars, S.D. DMH treatment significantly increased theproliferative zone metaphase index compared to non-DMH treatment in ratsmaintained on the p.o. solid and the p.o. liquid feeding regimens (p < 0.001) butnot in rats on the TPN feeding regimen. NS, not significant.

Table 5Crypt column height in the rat distal ileum

Rats were given 8 weekly injections of either DMH or vehicle. One week after the last injection, rats were placed onone of 3 different isocaloric feeding regimens for 10 days. Rats were killed 3 hr after receiving an i.p. injection ofcolchicine (1 mg/kg body weight). Values for each epithelial parameter were subjected to 2-way analysis of variance.

Crypt column ht(mm)Feeding

regimenSolid

p.o.Liquid p.o.TPNTreatmentNon-DMH0.216

Â±0.037a (14)"

0.197 Â±0.030 (12)0.191 Â±0.028 (5)DMH0.214

Â±0.043(16)0.205 Â±0.018(13)0.193 Â±0.032 (5)Row

Mean0.215

0.2010.192Crypt

column ht (no. ofcells)TreatmentNon-DMH35.64

Â±3.9830.83 Â±3.2830.41 Â±3.44DMH34.11

Â±5.7629.53 Â±1.4131.46 Â±2.68Row

Mean34.82

30.1530.94

Column mean 0.205 0.207 32.93 31.97



2.120.16PNSC

NSNSF1.13

10.420.42PNS

<0.001NS

Mean Â±S.D.6 Numbers in parentheses, number of rats.c NS, not significant.

MARCH 1983

1157




Table 6Crypt proliferative parameters in the rat distal ileum

Rats were given 8 weekly injections of either I>MH or vehicle. One week after the last injection rats were placed onone of 3 different isocaloric feeding regimens for 10 days. Rats were killed 3 hr after receiving an i.p. injection ofcolchicine (1 mg/kg body weight). Values for each epithelial parameter were subjected to 2-way analysis of variance.

No. of metaphase figures/crypt Crypt metaphase index

TreatmentFeeding

regimenSolid

P.O.Liquid p.o.TPNNon-DMH14.03

Â±3.43a (14)6

7.42 Â±1.48 (12)6.09 Â±1.57 (5)DMH13.93

Â±2.82(16)6.97 Â±1.02(13)6.88 Â±1.61 (5)Row

Mean13.98


Â±4.8812.16 Â±2.69

9.94 Â±2.19DMH20.89

Â±5.5411.79 Â±1.4510.88 Â±2.16Row

Mean20.41

11.9710.41

Column mean 10.19 10.23 15.28 15.94



71.110.24PNSC

<0.001NSF0.24

41.690.23PNS

<0.001NS

a Mean Â±S.D.b Numbers in parentheses, number of rats.0 NS. not significant.

Table 7

Metaphase index in the ear epidermis of the rat

Rats were given 8 weekly injections of either DMH or vehicle. One week afterthe last injection, rats were placed on one of 3 different isocaloric feedingregimens for 10 days. Rats were killed 3 hr after receiving an i.p. injection ofcolchicine (1 mg/kg body weight). Values for each epithelial parameter weresubjected to 2-way analysis of variance.

Metaphase index of ear epidermis

TreatmentFeeding

regimenSolid

p.o.Liquid p.o.TPNColumn

meanNon-DMH1.26

Â±0.24a (14)b

1.24 Â±0.11 (12)1.27 Â±0.21(5)1.25DMH1.19

Â±0.14(16)1.14 Â±0.18(13)1.1 2 Â±0.1 5(5)1.16mean1.22

1.191.202-way

analysis of variancestatisticsTreatment


0.320.21P<0.05

NSC

NSa Mean Â±S.D.6 Numbers in parentheses, number of rats.c NS, not significant.

observed in segments of colon transposed to the mid-small

intestine but not in the segments of small intestine exposed tothe milieu of the colon. These studies indicate that part of thedifference in response of these 2 tissues to chemical carcinogens is due to inherent factors within the colonie epithelium,perhaps related to DMA repair ability.

In past studies (46, 74) and in the present study, it was foundthat weekly DMH treatments enhance epithelial cell proliferation in the rat descending colon. The results of previous studiesusing mice (23, 65) and rats (46, 74) show that DMH treatmentleads to increased colonie crypt cell proliferation, colonie cryptproliferative zone size, and colonie crypt cellularity. Theseearly changes are observed prior to the appearance of focalatypias, which precede overt tumor formation in the mouse(22, 72) and the rat (46, 74). The present study is designed totest the differential susceptibility of rat colonie epithelium toDMH enhancement of cell proliferation compared to other

rapidly renewing cell populations, i.e., Â¡lealepithelium and earepidermis. The results show that DMH has a specificity ofstimulatory action for rat colonie epithelial cell proliferation andcrypt cellularity at the dosage of 9.5 mg/kg body weight.Epithelial cell proliferation and crypt cellularity in the rat distalileum were not influenced by DMH treatment.

There was a significant main effect of treatment on the ratear epidermis, although it was the opposite of the main effectof treatment found on colonie epithelium. DMH caused a significant decrease in the metaphase index of the ear epidermiscompared to non-DMH treatment. This may be an indirectresponse of the ear to the DMH-induced perturbance of the

epithelial cells in the descending colon and the response ofthese perturbed cells to the different feeding regimens, whichmay indirectly affect the nutritional status of the rat and thusaffect the mitotic activity of the ear epidermis.

Effects of Feeding Regimens on Epithelial Cell Proliferation and Crypt Cellularity in the Rat Descending Colon andDistal Ileum. Many previous investigators have examined theinfluence of nutrition on the epithelial kinetics of the rat proximalalimentary tract, but few have examined the influence of thetype, the level, and the route of administration of nutrition onepithelial kinetics of the rat colon (28, 37). Past studies indicatethat the lack of p.o. food intake causes intestinal atrophy anda reduced rate of cell renewal in the rat small (1, 2, 38, 41, 60,70) and large (40, 50, 71) intestines.

The present study is designed to test the influence of thephysical form, the chemical composition, and the route ofadministration of nutrition on epithelial cell proliferation andcrypt cellularity in the rat descending colon and distal ileum,while holding the caloric intake constant. The solid feedingregimen and the liquid feeding regimen differ in physical formand chemical composition. The solid feeding regimen and theTPN feeding regimen differ in physical form, chemical composition, and route of administration. The liquid feeding regimenand the TPN feeding regimen differ only in route of administration. The significant effects of feeding regimen on epithelial cellproliferation in the rat descending colon are discussed in thesection on interactions between DMH treatment and feeding





Table 8

Effects of recovery time from DMH treatment on crypt column height in the rat descending colon

Rats were given 8 weekly injections of DMH and killed 2, 4, or 8 weeks after the last DMH Injection. Rats were killed3 hr after receiving an i.p. injection of colchicine (1 mg/kg body weight). Values for each epithelial parameter weresubjected to 2-way analysis of variance.

Crypt column ht (mm) Crypt column ht (no. of cells)

TreatmentRecovery

time2

wk4wk8wkColumn

meanNon-DMH0.228

Â±0.025a0.213

Â±0.0180.221Â±0.0120.221DMH(8)6

0.234 Â±0.030(8)(6)

0.244 Â±0.027(5)(5)0.256 Â±0.042(4)0.242TreatmentMean

Non-DMH DMHMean0.231

32.820.22730.380.236

30.4831.43Â±

1.0034.84Â±0.5335.94Â±0.52 34.4435.07Â±

1.8433.83Â±1.3732.91Â±2.4432.242-way

analysis of variancestatisticsTreatmentRecovery

timeTreatmentXRecovery timeF6.850.420.13P<0.01NSCNSF64.072.700.57P<0.001NSNS

a Mean Â±S.D.

Numbers in parentheses, number of rats.0 NS, not significant.

Table 9

Effects of recovery time from DMH treatment on epithelial cell proliferation in the rat descending colon

Rats were given 8 weekly injections of DMH and killed 2. 4, or 8 weeks after the last DMH injection. Rats were killed3 hr after receiving an i.p. injection of colchicine (1 mg/kg body weight). Values for each epithelial parameter weresubjected to 2-way analysis of variance.

No. of metaphase figures/crypt Crypt metaphase index

TreatmentRecovery

time2

wk4wk8wkColumn

meanNon-DMH3.292.903.023.10Â±

0.88a(8)"Â±

0.41(6)Â±0.52 (5)DMH6.24

Â±2.28(8)4.90Â±1.94(5)4.95Â±1.45(4)5.54TreatmentMean

Non-DMH DMHMean4.77

5.013.814.813.88

5.054.96Â±

1.338.92Â±0.776.85Â±0.94 7.267.92Â±

3.256.97Â±2.765.73Â±2.276.032-way

analysis of variancestatisticsTreatmentRecovery

timeTreatmentx Recovery timeF20.471.180.05P<0.001NSCNSF13.470.840.08P<0.01NSNS

Mean Â±S.D.Numbers in parentheses, number of rats.

c NS, not significant.

regimen, because significant differences due to feeding regimen are observed only in DMH-treated rats.

Regardless of the route used for administration (p.o. or i.V.),the liquid elemental diet significantly suppresses colonie cryptcellularity compared to the solid feeding regimen across bothDMH and non-DMH treatments. The solid diet not only differs

in chemical composition from the liquid diet, but it providesnonabsorbable bulk. The reduction of nonnutritive bulk passingthrough the colon and/or differing chemical composition of thefeces are therefore implicated as factors in the suppressiveeffect of the liquid diets on crypt cellularity in both DMH- andnon-DMH-treated rats.

Because the diets given in the present study were isocaloric,any significant effects of feeding regimen on Â¡lealepitheliumcan be attributed to either the physical form of the diets, thechemical constituents of the diets, the route of administrationof the diets, or a combination of these factors. The liquidfeeding regimen and the TPN feeding regimen cause a significant suppression in epithelial cell proliferation and crypt cellularity in the rat ileum compared to the solid feeding regimen.

Regardless of the route of administration of the liquid diet, asignificant decrease in epithelial cell proliferation and cryptcellularity is seen; therefore, the ileal changes can be attributedto the physical form and/or the chemical constituents of thediet and not the route of administration.

In summary, epithelial cell proliferation and crypt cellularityin the rat distal ileum and crypt cellularity in the rat descendingcolon are suppressed by a liquid diet regardless of route usedfor administration (p.o. or i.V.). This suppression is due to thephysical form and/or the chemical constituents of the liquiddiet. Epithelial cell proliferation in the descending colon wassignificantly influenced by an interaction between DMH treatment and feeding regimen.

Effects of Interactions between DMH Treatment and Feeding Regimen on Colonie Epithelial Cell Proliferation. Experimental designs using animal models are important because ofthe increasing incidence of human large-bowel cancer in theUnited States. A number of investigators have demonstratedthat the level of dietary fat controls the eventual developmentof colon cancer in animal models (4-6, 19, 52, 61 ). Several

MARCH 1983 1159




previous studies (17, 30, 32, 78, 82) have reported that wheatbran has a protective effect against colon cancer, althoughother investigators (20) claim that bran does not have a protective effect on DMH-induced tumorigenesis in rats. A high intake

of carrageenan increases the evidence of colon cancer whileincreasing the intestinal and fecal bile acid levels (79). Possibleexplanations for these conflicting results are the following: (1 )variation in fibrous subcomponents between fiber types (21,31 ); (2) variations in the composition and relative concentrationof the other dietary components used by each investigator(55); and/or, (3) variations in the amount of DMH used by eachinvestigator. These previous studies have shown a relationshipbetween different nutritional factors and the incidence of induced tumors in animal models. The possible interaction between DMH treatment and different nutritional factors on epithelial cell proliferation in the rat colon can help in understanding the development of large-bowel cancer.

It appears that no studies have been designed to detect aninteraction between DMH treatment and feeding regimens onepithelial cell proliferation in the rat colon. Such an interactionwould test the effect of DMH treatment and feeding regimensoperating together on rat colonie epithelial proliferation. Oneway analysis of variance, which is used widely in biomedicairesearch, is not capable of detecting interactions betweenindependent variables. Thus, 2-way factorial analysis of variance is used in the present research design because a 2-way

analysis of variance is more parsimonious, more powerful, andable to detect the existence of an interaction between the 2independent variables (39).

Epithelial cell proliferation in the rat colon is significantlyinfluenced by an interaction between treatment and feedingregimen. A comparison of each type of treatment for eachseparate feeding regimen indicates that significant differencesdue to feeding regimen are observed only in DMH-treated rats.Non-DMH-treated rats do not show a differential response to

the various feeding regimens.The p.o. administration of the solid and liquid diets, regard

less of chemical constituents, supports the DMH-induced increases in colonie epithelial cell proliferation. TPN prevents theexpression of a DMH-induced increase in epithelial cell prolif

eration. This differential influence of feeding regimens on cellproliferation in DMH-perturbed epithelium is attributed to the

i.v. administration of the liquid diet compared to the p.o. administration of the liquid diet or the solid diet. Thus, the routeof administration has a significant influence on colonie epithelialcell proliferation in DMH-treated rats.

Irreversibility of DMH-induced Changes in Rat Colonie

Epithelium. Toth era/. (73) and Deschner and Long (24) foundthat whatever alterations are induced by DMH early in treatment, the changes are sufficient for induction of tumors whenadequate time for tumor development is given. Richards (66)has gone further in testing the reversibility of DMH-inducedchanges in mouse colonie epithelium. He found that the totalnumber of cells and the number of [3H]thymidine-labeled cells

in colonie crypts fluctuate during treatment with DMH and thesubsequent recovery period without DMH treatment. Whenmice are given 8 weekly injections of DMH, no overt tumorsare found after a 1-week recovery period, but 5 of 7 mice haveovert tumors after a 52-week recovery period. After 52 weeksof recovery, DMH-treated mice still have a significantly higher

number of cells per crypt column than do the control mice.

Maskens (49) has shown that new tumors continue to develop in the rat colon long after cessation of the immediatemetabolic and biological effects of DMH in the mucosa, andlong after the delay needed for carcinomas to grow from onecell to macroscopic size. All rats receiving one injection ofDMH will develop overt colonie adenocarcinomas if the latencyperiod is long enough (49). Thus, a differential rate of tumorinduction based on dosage and length of exposure to DMH andlatency period after DMH treatment has been demonstrated,as well as an irreversible state of change in colonie epithelium(24, 49, 66, 73).

The present study verifies the irreversibility of DMH-inducedeffects on the rat colonie epithelium following an 8-week recovery period.

Evidence for the Induction of a Preneoplastic State Produced by DMH in Rat Colonie Epithelium. Preneoplasia hasbeen defined as a heritable or irreversible state in which cellsshow an altered proliferative response to regulatory controls.The data from the present study support the concept that 8weekly injections of DMH at a dosage of 9.5 mg/kg bodyweight induces an irreversible preneoplastic state in the ratdescending colon, as measured by an 8-week recovery period.

Particularly revealing is the finding that significant changesin crypt kinetic parameters, induced by DMH treatment, do notrevert to control values following a 2- to 8-week recoveryperiod. An 8-week recovery period allows time for approxi

mately 14 to 28 complete renewals of the colonie epithelium,and yet the DMH-produced changes still persist. Based on an

altered and irreversible state of colonie epithelial cell proliferation induced by DMH, it is concluded that: (a) the preneoplastic state is a committed state and is not dependent upon thecontinued presence of the carcinogen; and (b) all cryptaiepithelium is preneoplastic, although not all cells progress tothe overtly transformed state. In addition, TPN prevents theexpression of this DMH-induced preneoplastic state of altered

epithelial cell proliferation.Based on experimental data and the biochemical properties

of DMH and related compounds, Maskens (47) has proposeda 2-step model of carcinogenesis. Analyses of the relationship

between dosage, number of cells at risk, time until tumor yield,along with previous studies on the etiology of DMH-inducedtumors, indicate that overt transformation involves 2 distinctand essential changes in DMH-perturbed cells (49). The firststep is caused by the carcinogen. This change is additive whensimilar doses are repeated and is transmissible within therenewing epithelium for prolonged periods. The results fromthe present study indicate that perturbed cells which haveundergone the first change do indeed have a proliferativeadvantage as compared to normal colonie cells, except whenrats are maintained on TPN. When subjected to different iso-calorie feeding regimens, DMH-perturbed colonie epithelial cell

proliferation responds significantly, whereas a significant response is not observed in normal colonie epithelial cell proliferation.

Carcinogen exposure is not required for the second step tooccur, although Maskens (49) indicated that continued presence of the carcinogen probably contributes to this change.The second step randomly affects DMH-perturbed cells whichhave undergone the first change. This second step seems tooccur at a constant rate within the rat colon (49). Support forthe proposed second step involved in DMH carcinogenesis is





the following: (a) the development of overt tumors in 70% ofanimals allowed a 52-week recovery period, following 8 weekly

DMH injections (66); and (b) the development of overt tumorswill occur in all rats receiving a single injection of DMH, if thelatency period is long enough (49).

The present study has used weekly DMH injections to inducethe first step, preneoplasia, in rat colonie epithelium. In doingso, an exploitable preneoplastic model has been established.This study is unique, because no other study has examined theinfluence of diet on cell proliferative parameters in DMH-in-

duced preneoplastic colonie crypts. The ability to manipulatedietary factors and to examine their influence on this preneoplastic model can help define the role that dietary regimensplay in promoting or preventing the progression of preneoplastic cryptai epithelium toward overt transformation.

Additional investigation is under way to verify this preneoplastic system and to elucidate the possible interaction between DMH treatment and level of caloric intake, when theroute of administration (i.v.) and chemical composition of a dietare held constant.

ACKNOWLEDGMENTS

The authors wish to thank Ginny Ord, Carol Harmison, and Steve Dalley fortheir technical assistance, and Lorraine Babcock, Monny Sklov, and RobertWood for their assistance with the statistical analysis used in this study. Theauthors also wish to thank Cathy Miller and Betty Russell for their careful typingof this manuscript.

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1983;43:1153-1162. Cancer Res David W. Heitman, Barry G. Grubbs, Teri O. Heitman, et al. Regimen on Rat Colonic Epithelial Cell ProliferationEffects of 1,2-Dimethylhydrazine Treatment and Feeding

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Effects of 1,2-Dimethylhydrazine Treatment and Feeding ......Sixty-five male Sprague-Dawley rats 4 to 6 weeks old (mean weight, 112 g; Simonsen, Gilroy, Calif.) were divided randomly