biochemical and clinical effects of selenium on - cancer research

[CANCER RESEARCH 41, 4458-4465. November 1981]0008-5472/81/0041 -OOOOS02.00

Biochemical and Clinical Effects of Selenium on Dimethylhydrazine-inducedColon Cancer in Rats1

Maryce M. Jacobs,2 Curtis F. Forst, and Faith A. Beams

The Eppley Institute for Research in Cancer. University of Nebraska Medical Center. Omaha, Nebraska 68105

ABSTRACT

The biochemical and clinical effects of selenium (Na2SeO3)on 1,2-dimethylhydrazine (DMH)-induced colon carcinogene-sis in male Sprague-Dawley rats are presented. A 4-ppm se

lenium supplement to the drinking water was provided before,during, and after 20 weekly injections of 20 mg DM H per kgbody weight. Immediately after the 20th DMH injection, part ofthe rats were sacrificed. The incidences of colon tumors ingroups provided selenium before DMH, before and duringDMH, and only during DMH treatment were reduced to 39, 43,and 36%, respectively. The incidence in the DMH only controlwas 63%. Other rats in all treated and control groups weremaintained up to 5 months post-DMH treatment. At 10-week

intervals throughout the study, selected blood and tissue components were analyzed. The following hematological changescorrelated with DMH treatment, (a) Serum glutamic oxalacetictransaminase increased 2-fold (normal, 66 Â± 14 g/dl). (o)

Serum alkaline phosphatase increased 24% (normal, 166 Â±56 units/liter), (c) Serum protein decreased 14% (normal, 6.77Â± 0.48 g/dl). (d) White blood count increased 2- to 3-fold(normal, 7.7 Â± 2.7 x 103/cu mm). And (e) hemoglobin de

creased 67% (normal, 18.1 Â± 1.3 g/dl). The magnitude ofthese changes varies with each selenium treatment group andwith each 10-week analysis period. Provision of 4 ppm sele

nium doubled both liver and blood selenium levels comparedto unsupplemented controls. The effects of selenium and DMHtreatments on glutathione peroxidase and /i-glucuronidase ac

tivities and on sialic acid are presented. Possible mechanismsby which selenium protects against DMH-induced neoplasia

are discussed.

INTRODUCTION

Previous studies have demonstrated selenium inhibition ofcolon, liver, skin, and mammary gland carcinogenesis. Usingthe male Sprague-Dawley rat model, selenium inhibited thecolon tumor incidence in DMH3-treated rats and reduced the

total number of colon tumors in MAM-treated rats (14, 17).Selenium inhibited 2-acetylaminofluorene (25) and 3'-methyl-

4-dimethylaminoazobenzene (11) hepatocarcinogenesis. Thehepatic tumor incidence induced by 3'-methyl-4-dimethylami-

noazobenzene was reduced by both inorganic selenium(Na2SeO3) and by organic selenium (selenium yeast) supplements to the drinking water and diet, respectively. Reportsfrom other laboratories have noted that selenium supplements

' Supported by the National Large Bowel Cancer Project Grant R26 CA 25699

of the National Cancer Institute.* To whom requests for reprints should be addressed.3 The abbreviations used are: DMH. 1,2-dimethylhydrazine; MAM, methylaz-

oxymethanol; CBC. complete blood count; SGOT, serum glutamic oxalacetictransaminase; GSHP. glutathione peroxidase.

Received March 27, 1981 ; accepted August 11, 1981.

decreased the incidence of hepatic tumors induced by 3'-

methyl-4-dimethylaminoazobenzene (4) and 2-acetylaminfluo-

rene (12) and of skin (31 ) and mammary tumors (13) inducedby 7,12-dimethylbenz(a)anthracene. Selenium added to the

drinking water reduced the incidence of spontaneous mammary tumors in C3H/St mice (29).

Epidemiological studies illustrate an increased incidence ofcolorectal, breast, and other cancers in humans in geographicregions where selenium is deficient (19, 33). Lower bloodselenium levels in patients with gastrointestinal cancers havebeen observed in several clinical studies (1, 32). In experimental systems, supplements of selenium reduced the incidence ofcolon, breast, and liver cancers as cited above. In additionalstudies, we have reported the comparative reduction in DMH-

induced colon tumors by supplements of selenium to drinkingwater, ascorbic acid to the diet, and butylated hydroxytolueneto the diet (16). In this study, selenium is provided before,during, and after DMH treatment. The "parameters" evaluated

throughout include the CBC, serum protein, alkaline phosphatase, and SGOT. Liver selenium and GSHP, a selenium-de

pendent enzyme, are correlated with microscopic pathology,SGOT, and alkaline phosphatase levels to insure that theselenium level added to the drinking water is not toxic. Thecolonie /i-glucuronidase activity is assayed as a possible indi

cator of tumor development. The potential preventive and therapeutic attributes of selenium in colon cancer and the possiblemechanism of action of selenium are discussed.

MATERIALS AND METHODS

Weanling male Sprague-Dawley rats purchased from SASCO

(Omaha, Nebr.) were quarantined, randomized into treatment andcontrol groups, and earmarked. Animals were housed 5/cage in plasticcages containing granular cellulose bedding (Bed-o'-cobs; Anderson

Mills, Mony. Ohio) in temperature- and humidity-controlled rooms. Abasal diet of Wayne Lab-Blox was provided ad libitum. Stock solutions

(400 ppm) of selenium (Na2SeO3; purchased from Alfa Products,VentrÃ³n Corp., Danvers, Mass.) were prepared weekly, diluted to 4-

ppm selenium concentrations, and provided ad libitum to appropriategroups for defined time intervals during the study outlined below. TheWayne diet varies in composition between 0.1 and 0.5 ppm seleniumand the drinking water contains less than 10 ppb selenium. Accordingto our assay, the Wayne diet used in this study was 0.183 ppmselenium. Hence, our 4-ppm Se supplement was 22-fold higher than

the dietary level in untreated controls. Rats treated with carcinogenreceived weekly s.c. injections of 20 mg of DMH (sym-dimethylhydra-

zine dihydrochloride; 97%; purchased from Aldrich Chemical Co. Inc.,Milwaukee, Wis.) per kg body weight for 20 weeks. Body weights andsurvival were monitored throughout. At either death or scheduledsacrifice, complete necropsies were performed for evaluation of grossand microscopic pathology. Colons were resected at the cecal andrectal junction and cut longitudinally. One half was preserved in formalin and hematoxylin and eosin stained for microscopic pathology.The other half of the colon was quick frozen on dry ice or rinsed in

4458 CANCER RESEARCH VOL. 41

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Selenium Inhibition of DMH-induced Colon Cancer

water and immediately scraped with a microscope slide for colonmucosa assays such as /8-glucuronidase (27).

For clinical analysis and CBC, animals were anesthetized by CO2inhalation and exsanguinated by heart puncture. SCOT, alkaline phos-

phatase, and protein were determined on the Polimak Model 900automated batch analyzer (P.M. America, Inc., Sacramento, Calif.). ForSGOT, the AST (glutamic oxalacetic transaminase) Uni-UV diagnostic

kit purchased from Sciavo, Inc. (Wayne, N. J.) was used. This is anautomated kinetic assay in which the rate of NADH oxidation is proportional to the SGOT activity. For the alkaline phosphatase assay, theALP-cinet diagnostic kit purchased from Sciavo, Inc., was used. Thisautomated procedure uses p-nitrophenylphosphate and is an end pointassay for p-nitrophenolate. The reagent kit for total protein was pur

chased from Coulter Electronics, Inc. (Hialeah, Fla.) and uses a modified Biuret method. For confirmation, the manual assay of Lowry ef al.(24) was carried out for protein and the method of Nyc et al. (26) foralkaline phosphatase. The CBC was determined on the Coulter ModelZF-5 and the hemoglobinometer both from Coulter Electronics, Inc.

These analyses were carried out on freshly drawn whole blood orserum.

Sections of liver removed at necropsy were quick frozen on dry iceand stored frozen until homogenized and assayed. Selected manualassays were carried out on blood, liver, or colonie scrapings. Liver andblood levels of selenium were measured by the spectrofluorometricmethod of Watkinson (35) using an Aminco-Bowman spectrofluorom-

eter. GSHP was assayed according to Burk ef al. (2) using bothhydrogen peroxide and eumene hydroperoxide as substrates. Othermanual assays were for/3-glucuronidase (27), sialic acid (34), and acidphosphatase (18). The Gilford Model 250 UV-VIS spectrophotometer

was used for these assays. Linear regression and other statistics werecalculated as described by Colton (5).

All rats were initiated on the study at 8 weeks of age. All DMHinjections were given between 21 (first injection) and 40 (last injection)weeks of age. Relative to DMH treatments, supplemental selenium wasprovided in the drinking water for designated periods (weeks of age)either before, during, after, or combinations thereof. The experimentalgroups are as follows: Group 1, selenium given only during DMHtreatment (21 to 40 weeks of age); Group 2, selenium given only for13 weeks before DMH treatment (8 to 21 weeks of age); Group 3,selenium given for 13 weeks before and during DMH treatment (8 to40 weeks of age); Group 4, selenium given only DMH treatment (41weeks of age to death); and Group 5, selenium given during and afterDMH treatment (21 weeks of age to death). The control groups wereas follows: Group 6, selenium only (8 weeks of age to death); Group 9,DMH only; and Group 10, untreated control. Two additional seleniumonly control groups were run: Group 8, selenium only (21 weeks ofage to death); and Group 8, selenium only (41 weeks of age to death).Because the body weight, survival, and all biochemical and clinicalparameters measured were identical in Groups 6 to 8 selenium only

controls, only the data for Group 6 are presented in the tables andtext. When selenium supplements were not provided, tap water was

available ad libitum.

RESULTS

Survival and Growth. Table 1 presents the survival and bodyweight for treatment and control groups at 10-week intervalsthrough 61 weeks of age. The 4-ppm selenium supplement had

no effect on survival. At 61 weeks of age, the survival of theselenium only control (Group 6) and of the untreated control(Group 10) was equivalent, 98 and 96%, respectively. In contrast, the survival of all DMH-treated groups was less than 50%

at 61 weeks of age. At 41 weeks of age, the survival of allgroups was 96% or greater. This was 1 week following the20th DMH injection. Ten and 20 weeks following DMH treatments, the survival rate was markedly reduced compared toselenium only and untreated control groups. The survival ofGroup 5 rats given selenium during and after DMH was poorestand most notably different from all other groups (67% at 51weeks and 30% at 61 weeks). The Group 2 rats providedselenium only before DMH had better overall survival than otherselenium- and DMH-treated groups (Groups 3 to 5).

Table 1 also shows the body weight of experimental groupsat 10-week intervals. Initially, all groups were weight matchedat 106 Â±12 g/rat. The selenium only control (Group 6) weightgain at 61 weeks of age was slightly less than the untreatedcontrol, but with the overlap of standard deviation betweenthese 2 groups this difference was not significant. During the20 weeks of DMH treatment (Table 1,21 to 41 weeks of age),the selenium only and untreated control groups gained approximately 67 g, the DMH only groups gained 45 g, and the DMHand selenium given together groups gained 4 g, suggesting apossible antagonism between DMH and selenium. By comparison, provision of selenium before DMH (Group 2) resulted ina 47-g weight gain, implying partial protection by seleniumagainst DMH. Over the weeks following the last DMH injection(41 to 61 weeks of age), the weight responses were as follows:Group 2, -9 g; Group 3, +16 g; Group 4, -57 g; Group 5,+ 9 g; and Group 9, -9 g. Early and prolonged treatment with

selenium protected against weight loss. Provision of seleniumonly after DMH treatment (Group 4, -57 g) was detrimental

causing considerable weight loss.Colon Tumors. For colon tumors, Table 2 shows the inci

dence, mean diameter, total number per group, and number of

Table 1

Effects of selenium on survival and body weight of rats treated with DMH

Group1

234569

10TreatmentDMH

+ seleniumSelenium before DMHSelenium before and during DMHSelenium after DMHSelenium during and after DMHSelenium onlyDMH onlyUntreated control21%

of survival100

(60)c

100(120)98 (120)

100(120)100 (80)100 (120)100 (75)100 (60)wk"BW"(g)458

Â±40d

442 Â±42435 Â±42451 Â±42455 Â±47441 Â±42443 Â±45461 Â±4231%of

survival97

1000888

10089

10098wk"BW(g)454

Â±41476 Â±49453 Â±47476 Â±45453 Â±49485 Â±45470 Â±44508 Â±4841%of

survival97

97969799989898wk"BW(g)462

Â±49489 Â±51467 Â±47493 Â±48478 Â±54506 Â±45488 Â±42530 Â±5351%ofsurvival83

768967988697wk"BW(g)531

Â±57513Â±53

476 Â±60482 Â±63521 Â±49526 Â±51572 Â±5261%ofsurvival43

363430

984796wk'BW(g)480

Â± 71483 Â± 63436 Â±109487 Â± 73533 Â± 50517 Â± 67580 Â± 54

" 21 weeks old, begin DMH; 31 weeks old, after 10 weeks of DMH; 41 weeks old.

last DMH injection; 61 weeks old. 20 weeks following last DMH injection.* BW, body weight.' Numbers in parentheses, initial number of rats per group." Mean Â±S.D.

after 20 weeks of DMH (final DMH treatment); 51 weeks old. 10 weeks following

NOVEMBER 1981 4459



M. M. Jacobs et al.

Table 2

Colon tumor incidence during and after 20 weekly injections of DMH with and without supplementary selenium31wk"Group123456910TreatmentDMH

+seleniumSeleniumbeforeDMHSelenium

beforeandduringDMHSelenium

afterDMHSeleniumduringandafter

DMHSeleniumonlycontrolDMH

onlycontrolUntreatedcontrolIncidence(%)27

(8/30)c6(1/16)20(3/15)12(2/16)27

(8/30)0(0/15)12(2/16)0

(0/9)Mean

diameter(mm)3.22.03.04.73.24.7Totaltumors913393T/TBA*1.11.01.01.51.11.5Incidence(%)36(10/28)39

(11/28)43(13/30)63(19/30)36(10/28)0

(0/30)63(19/30)0

(0/10)41

wk"Mean

diameter(mm)3.84.54.15.63.85.6Totaltumors291728312931T/TBA2.91.52.11.62.91.6Incidence(%)85(61/72)91

(62/68)91

(63/69)87(59/68)0

(0/15)91(29/32)0

(0/16)51

to61Mean

diameter(mm)7.77.57.26.06.7wkaTotaltumors17518222216574T/TBA2.92.93.52.82.6

At 31 weeks of age, 10 weekly injections of DMH have been administered; at 41 weeks of age, 20 weekly injections of DMH have been administered; 51 to 61weeks of age corresponds to 10 to 20 weeks following the last DMH injection.

6 T/TBA, colon tumors per tumor-bearing animal.1 Numbers in parentheses, number of animals with colon tumors per total number of animals examined per group.

colon tumors per tumor-bearing animal per group. These data

are itemized for treatment and control groups midway throughDMH injections (31 weeks of age), immediately after the fullcourse of 20 DMH injections (41 weeks), and 10 to 20 weeksfollowing the last DMH injection (51 to 61 weeks). Tumors wereverified histopathologically to be adenocarcinomas and villousadenomas of the colon. Colon tumors often developed out ofPeyer's patches approximately 5 cm from the cecal junction

and 2 to 5 cm from the rectal junction.Immediately after the 20th DMH injection, 30 rats were

sacrificed at 41 weeks of age in each experimental group and10 rats in the untreated control. Provision of selenium simultaneously with DMH treatment reduced the colon tumor incidence from 63% (Group 9; DMH only control; 41 weeks old) to36% (Group 1, 41 weeks old). This 50% reduction confirmedearlier reports from this laboratory (14, 16, 17).

Selenium provided before and during (Group 3) as well asonly before (Group 2) DMH treatment also reduced the colontumor incidence to 43 and 39%, respectively, compared to theDMH only control of 63% (Table 2, 41 weeks old). Between 51and 61 weeks of age or following 10 to 20 weeks from the lastDMH injection, all DMH-treated groups developed an approxi

mate 90% colon tumor incidence regardless of whether theselenium provision was before, during, and/or after carcinogenadministration.

The majority of DMH-induced colon tumors were roughly

spherical and symmetrical. The mean diameter and tumorvolume are only crude estimates but do indicate the relativedegree of tumor burden in various treatment groups. In general,the mean diameter increased with time from means of 3.2 (31weeks of age; tenth DMH injection) to 4.5 (41 weeks of age;20th DMH injection) and to 7.0 mm (51 to 61 weeks of age; 10to 20 weeks following the 20th DMH injection).

Considering all of the data in Table 2, 3 observations areoutstanding, (a) At 31 weeks, Group 2 rats provided seleniumonly before DMH had the lowest tumor incidence, lowest meantumor diameter, and lowest number of tumors per tumor-bear

ing animal, (b) At 41 weeks, Group 1 rats provided seleniumonly during DMH treatment had the smallest tumor diameterand lowest tumor incidence. And (c) between 51 and 61 weeks,Group 5 rats given selenium during and after DMH had thesmallest tumor diameter and lowest number of tumors per

tumor-bearing animal among selenium- and DMH-treated

groups. In summary, selenium supplementation before DMHtreatment protected against the short-term effects of DMH,

while selenium given during and after DMH offered protectionagainst long-term DMH-induced tumorigenesis. Provision of

selenium only after DMH treatment (Group 4) appeared toenhance the development of new colon tumors since at 41weeks of age there were 1.6 tumors/tumor-bearing animal andthis increased to 3.5 tumors/tumor-bearing animal between 51

and 61 weeks of age.Extracolonic tumors were not apparent in animals sacrificed

at intervals up to 41 weeks of age corresponding to the 20thDMH injection. After this time, however, tumors were observedin the small intestine (40%), pancreas (35%), Zimbal gland(38%), and cecum (2%) with the incidence unchanged inselenium-supplemented animals compared to DMH only controls. Presumably, these tumors are primary tumors at extra-

colonie sites but may, in fact, represent metastasis. In allanimals having extracolonic tumors, there were also colontumors.

CBC. The CBC includes RBC, WBC, hematocrit, hemoglobin,and calculated mean cell volume run on individual animals.Table 3 shows the CBC for all groups at 41 weeks of age.Since the CBC was unchanged during tumor initiation and earlydevelopment compared to selenium only and untreated controlgroups, only the data from selected experimental groups at 41,56, and 64 weeks of age are presented to illustrate alterationsassociated with DMH treatment. The mean calculated cellvolume did not change with increased age or selenium with orwithout DMH treatment and is therefore reported only at 41weeks of age.

The WBC increased 2- to 3-fold with DMH treatment. The

increase observed in Groups 2 to 5 and 9 (DMH only control)at 56 weeks was returned closer to the normal WBC (7.7 X103/cu mm) at 64 weeks of age in Groups 2, 4, and 5. This

compares favorably with the earlier report in which selenocys-

tine treatment reduced total leukocyte counts providing therapeutic control of leukemia patients (39).

Comparing the hematocrit in selenium only and untreatedcontrol (Table 3, Group 10) groups, there is a decrease tosimilar levels with increased age. The decrease is also presentin DMH-treated groups (Groups 2 to 5) at 64 weeks of age.




Selen/um Inhibition of DMH-induced Colon Cancer

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enon that does not independently reflect either selenium orDMH treatment.

The hemoglobin decreases with increased age in seleniumonly (Table 3, Group 6), untreated control (Group 10), andDMH only (Group 9) groups between the ages of 41 to 64weeks. The decrease is greatest in DMH with or without selenium treatment groups and is sustained with time. The reduction in hemoglobin is not restored to normal by seleniumsupplementation either before or after DMH treatment.

Serum Protein, SCOT, and Alkaline Phosphatase. According to Table 4, there is an apparent reduction in the serumprotein level with age and a greater reduction with DMH treatment. Between 41 and 61 weeks of age, the mean serumprotein for Groups 2 to 5 declines 0.93 g/dl, the selenium onlyGroup 6 rises 0.17 g/dl, and the DMH only and untreatedcontrols decline 1.09 and 0.47 g/dl, respectively. Not only isthe serum protein level age related but also this parametercorrelates with DMH treatment.

The serum SGOT level was determined on individual animalssacrificed at increasing time intervals before, during, and afterDMH with and without selenium treatment. The SGOT activityat 41,51, and 61 weeks of age is expressed in Table 4. Largestandard deviations were characteristic throughout. Nevertheless, the SGOT increased markedly in DMH- and selenium-

treated Groups 2, 4, and 5 with increased age and progressivetumor development.

Also illustrated in Table 4 are the serum alkaline phosphataselevels in DMH with and without selenium and control animals at41, 51, and 61 weeks of age. As with SGOT, the standarddeviations are large in each group and increase with increasedtumor burden reflecting individual animal variation within thegroups. The baseline normal level was 166 Â± 56 units/literand remained as such within standard deviation up to 61 weeksof age in selenium only controls (Table 4, Group 6) and theuntreated control. All DMH-treated groups had markedly in

creased alkaline phosphatase levels compared to the untreatedcontrol at each 10-week interval assayed. The selenium only

control (Table 4, Group 6) had a higher serum alkaline phosphatase than the untreated control but lower than DMH-treated

groups.For each clinical test, the proposed increase or decrease in

response to treatment evolved from a comparative analysis ofdata obtained for treatment versus control groups with increasein age. This approach was taken because of the characteristically large variation among individual animals within a group.At a single time, assays were frequently not significantly different between treatment and control groups. It was thereforenecessary to trace the history of individual animals throughouttreatment from 8 to 64 weeks of age with respect to eachassay (e.g., alkaline phosphatase). The comparison of changesoccurring in one group with changes in another within anidentical time frame gave rise to the selection of markers whichappeared to correlate with colon tumor development and/orDMH treatment. For example, the alkaline phosphatase level at41 weeks of age in Group 4 is 219 Â±109 units/liter and is notsignificantly increased over the untreated control Group 10(127 Â±33 units/liter). The enzyme level in Table 4 for Group4 (selenium after DMH) at 10-week intervals is as follows: 21

weeks, 197 Â±51 units/liter; 31 weeks, 204 Â±29 units/liter;41 weeks, 219 Â±109 units/liter; 51 weeks, 246 Â±125 units/

NOVEMBER 1981 4461



M. M. Jacobs et al.

Table 4

Effect of selenium on serum protein, SGOT, and alkaline phosphatase in rats treated with DMH

Protein (g/dl) SGOT (unit/liter) Alkaline phosphatase (unit/liter)

Group Treatment 41 wk 51 wk 61 wk 41 wk 51 wk 61 wk 41 wk 51 wk 61 wk

123456910DMH+seleniumSeleniumbeforeDMHSelenium

beforeandduringDMHSelenium

afterDMHSeleniumduringandafter

DMHSeleniumonlycontrolDMH

onlycontrolUntreatedcontrolBaseline

at 21 wk of age6.176.296.076.136.176.576.136.276.77Â±

0.48aÂ±

0.66Â±0.38Â±

0.66Â±0.48Â±

0.49Â±0.38Â±0.66Â±0.485.59

Â±0.775.24Â±0.925.51

Â±0.845.66Â±1.057.01

Â±0.355.88Â±0.877.08Â±0.335.0

Â±0.695.09Â±0.785.35

Â±0.625.50Â±0.426.74

Â±0.275.04Â±0.675.80Â±0.2971

Â±2878Â±3365Â±1976

Â±3171Â±2855

Â±1476Â±3170Â±2766Â±1471

Â±1586Â±5288

Â±3486Â±3746

Â±988Â±3452Â±12140

Â±8576Â±27111

Â±S3118Â±1157

Â±3274Â±1957Â±11221

Â±93200Â±85218Â±84219

Â±109221Â±93165Â±

73219Â±1091

27 Â±33166Â± 56264

Â±132172Â±85246

Â±125229Â±76152

Â±61206Â±91202Â± 84231

Â±64203Â±94254

Â±137273Â±133171

Â±27241Â±116110

Â± 46

" Mean Â±S.D.

Table 5Effect of selenium on liver selenium and GSHP, serum acid phosphatase, and sialic acid and colon ÃŸ-glucuronidase with DMH treatment of Sprague-Dawley rats

Liver GSHP(Â»MNADP/mg

Group Treatmentprotein)31

DMH + selenium 60.8 Â±10.8rt*58.2Â±2.5g2

Selenium before DMH 65.2 Â±14.0e55.6Â±9.0"3

Selenium before and during DMH 64.9 Â±9.3*57.4Â±6.6"6

Selenium only 52.7 Â±11.4*53.6Â±6.5"9

DMH only 76.4 Â±17.2*59.5Â±10.6"10

Untreated control 107.9 Â±14.8*82.1Â±16.79Serum

acidphosphatase(Â»MNP6

hy-Liver

sele- drolyzedxnium(ug10"3/mgselenium/g

protein/20wet wt)min)2.36(21)'3.24(41)2.65(21)2.02(41)1.79(51)2.50(61)3.05(21)2.06(41)2.10(51)2.37(61)0.702

1.71(21)1.93(41)1.69(51)1.71

(11)2.60(21)2.37(41)2.07(51)2.08(61)0.445

2.01(21)2.55(41)2.40(51)2.24(61)Sialic

acid (Â«MNANAhydro-lyzedx10~3/mgprotein/30

min)Serumc26.6

Â±4.4(31)23.5Â±4.6(36)22.4Â±3.2(41)23.4Â±6.0(31)21.9Â±1.9(41)38.5Â±7.3(61)28.0

Â±8.1(31)33.1Â±6.5(41)26.3Â±2.3(51)40.4Â±6.4(61)20.0Â±4.0(31)28.7Â±2.5(41)28.9Â±6.4(61)19.1

Â±2.6(31)29.2Â±4.1(41)32.6Â±3.0(51)44.7Â±5.0(61)22.7Â±3.1(31)28.5Â±4.4(41)31.5Â±5.3(51)44.3Â±4.1 (61)Colonc7.1

(31)7.7(31)6.7(41)13.5(31)9.6(41)9.5(51)11.6(31)8.0(41)9.4(51)8.2(31)9.4(41)8.1

(51)9.6(31)10.4(41)10.6(51)Colon

/?-glucuronidase(mMphenolphthaleinglucuronidehydrolyzed/mg

protein/hr)Bacterial

Colonie0.115(41)0.079(41)0.843(41)

0.099(41)0.090(51)0.598(41)

0.114(41)0.070(51)0.693(41)

0.075(41)0.085(51)0.383(41)

0.084(41)0.065(51)

11All assays at 31 weeks of age following 10 injections of DMH." NP. p-nitrophenolate; NANA, W-acetylneurammic acid.1 Serum assays on 3 to 6 rats: colon assays on pooled sample from 2 to 5 rats." Mean Â±S.D.a Substrate, hydrogen peroxide.' Number in parentheses, weeks of age at time of analysis.3 Substrate, eumene hydroperoxide.

liter; 61 weeks, 254 Â± 137 units/liter. The correspondingenzyme levels in the untreated control Group 10 are: 21 weeks,197 Â± 51 units/liter; 31 weeks, 178 Â± 45 units/liter; 41weeks, 127 Â±33 units/liter; 51 weeks, 202 Â±84 units/liter;and 61 weeks, 110 Â±46 units/liter. Comparison of these andadditional data at intermediate time intervals between 8 and 61weeks of age suggests there is in fact an increase in alkalinephosphatase.

An alternate method of analysis was attempted to obtain amore direct correlation between tumors and each clinical response. This method was based on the observation that thecolon tumor incidence through 41 weeks of age was less than100% in DMH-treated groups. Each clinical test per groups

was subdivided into responses in animals with tumors versusresponses in animals without tumors. There were no differ

ences. This implied that early changes (initiation of DMH-in-

duced tumors) which develop into gross and microscopicallyidentifiable tumors had already occurred. This gave rise toclinical responses which precede morphological changes commonly used for pathological evaluation.

Selenium and GSHP. Using the spectrofluorometric methodof Watkinson (35), both liver and blood selenium levels weremeasured at 41 weeks of age. The untreated control Group 10(Table 5) had 0.445 jug selenium per g wet weight of liver, andthe selenium only control Group 6 had nearly 2-fold higher

selenium or 0.702 fig selenium per mg wet weight of liver.Similarly, the blood selenium levels at 41 weeks of age increased from 0.33 /Â¡gselenium per ml blood in the untreatedcontrol to 0.662 /ig selenium per ml blood in the selenium onlyGroup 6.





Liver obtained at necropsy of 41-week-old rats were quick

frozen on dry ice and kept frozen until GSHP analysis. Immediately before analysis, the livers were homogenized and cen-

trifuged, and the supernatant was assayed for GSHP activity(2). Using hydrogen peroxide and eumene hydroperoxide assubstrates, the units of GSHP activity are expressed as /IMNADPH oxidized per mg protein. Table 5 illustrates the GSHPactivity measured in corresponding animals with both substrates. The selenium only control exhibited the lowest activity(Group 6, 52.7 Â±11.4 units), the DMH- and selenium-treated

Groups 1 to 3 an intermediate level (mean, 63.6 units), and theuntreated control the highest level (107.9 Â±14.8 units). Similarly, the selenium-independent GSHP assayed with eumene

hydroperoxide was lowest in the selenium only control, intermediate in selenium and DMH Groups 1 to 3, and highest inthe untreated control. Having obtained these results with liversamples, pooled samples of colonie scrapings were assayedfor the selenium-dependent GSHP using H2O2 as substrate.The selenium only-supplemented Group 6 was 5.39 Â±1.2

units and was higher than the DMH control Group 9 (4.66 Â±1.5 units). Again, the untreated control Group 10 was highest(5.76 Â±2.7 units) and the DMH and selenium Group 3 was atan intermediate level of 4.85 Â± 0.9 units. These data aresupplementary to Table 5. A possible interpretation of theGSHP data is presented in "Discussion."

Earlier, we reported the acute and chronic toxicity of selenium using survival, growth, clinical chemistry, and pathologyas monitors (15). On the basis of these data, we selected 4ppm selenium as the supplemental dose for these carcinogen-esis studies. High (16- and 64-ppm) selenium stunted growth

and increased the serum alkaline phosphatase and SGOTlevels, while selenium supplements below 8 ppm had no effecton these parameters. Liver cell degeneration and necrosis, aswell as bile duct proliferation, were observed in occasionaluntreated control and high-dose (16- and 64-ppm) selenium-

treated rats. Neither pyknotic cells nor a large number ofdividing cells accompanied this observation, thus denouncingpremalignant tendencies related to high doses of selenium.The increase in SGOT in 16- and 64-ppm selenium-treated

groups is in clinical agreement with damage to the parenchymalcells but is not observed in 4-ppm selenium-treated groups.

None of the above monitors of selenium toxicity was observedin the 4-ppm selenium only control Group 6 through 69 weeks

of age. Morphological, clinical, and biochemical changes arein large part due to DMH treatment but also to increased ageand to the combined effects of selenium and DMH treatment.

/Â¿-Glucuronidase. Human and animal studies associate anincrease in fecal bacterial /?-glucuronidase with increased co

lon cancer or increased risk (10, 28). Using the same method(27) with an adjustment of the reaction mixture to pH 7.0 forthe bacterial enzyme and to pH 4.5 for the colonie enzyme, weassayed both enzyme sources. The units of /3-glucuronidaseactivity are expressed in Table 5 as rriM phenolphthalein glu-

curonide hydrolyzed per mg protein per hr. Both enzymes arelowest in the untreated control Group 10, increased in theselenium only control Group 6 and DMH only control Group 9,and appear to be highest in DMH and selenium Group 3 (Table5). The colonie enzyme is approximately 5 to 10 times lowerthan the bacterial enzyme. Both sources of the enzyme wereassayed at 41 weeks of age or 1 week following the 20th DMHinjection. The colonie enzyme was again assayed 10 weeks

later at 51 weeks of age.Sialic Acid. If in fact an increase in mucopolysaccharides

(containing sialic acid residues) in colon or serum were relatedto selenium and/or DMH treatment, supportive data from sialicacid assays were ascertained. The serum sialic acid determinations were after 10, 15, and 20 DMH injections and thecolonie sialic acid determinations following 10 and 20 DMHinjections. The sialic acid concentration is expressed in unitsas /IM W-acetylneuraminic acid hydrolyzed x 10~3 per mg

protein per 30 min in Table 5. The sialic acid level is approximately 4-fold higher in the serum than in the colonie scrapings.

With DMH treatment and colon tumor induction, the serumsialic acid increased. The colon sialic acid showed no apparentchange although these assays were conducted on pooledcolon scrapings from 2 to 5 rats/assay. Whether or not selenium supplementation prevents as large an increase as observed in the DMH only controls will require more assays forclarification. The data in Table 5 indicate that the increase inserum sialic acid is an age-related phenomenon.

Acid Phosphatase. The serum acid phosphatase activityincreases in prostatic carcinoma and multiple myeloma withconcurrent changes in isozyme patterns (9). Determination ofa possible correlation between colon carcinoma and the acidphosphatase was attempted. Table 5 shows the acid phosphatase expressed in units as /IM p-nitrophenyl phosphate hydrolyzed x 10~3 per mg protein per 20 min for treatment and

control groups. Individual serum samples from 5 to 20 rats/group were assayed at each 10-week interval. The mean acid

phosphatase in the untreated control Group 10 was 2.3 Â±0.23 units and was unchanged through 61 weeks of age. Withselenium supplementation, the mean level of 1.76 Â±0.11 units(Table 5, Group 6) was lower and remained constant withincreased age. All groups receiving DMH with or without theselenium supplement had higher acid phosphatase levels thanthe selenium only control but slightly less than the untreatedcontrol. The DMH and selenium treatment and colon tumordevelopment appeared to be accompanied by a reduced acidphosphatase level at 41 weeks of age (last DMH injection) andat 51 weeks of age (10 weeks after the last DMH injection) inGroups 2 and 3 (Table 5). The increased acid phosphataselevel in prostatic cancer and other diseases is commonly determined using /8-glycerophosphate as substrate. The inverseresponse using the p-nitrophenyl phosphate synthetic sub

strate in this study possibly reflects different substrate specificities of the acid phosphatase and a possible means of distinguishing prostatic from colonie cancer.

DISCUSSION

In the foregoing study, many clinical and biochemical assayshave been conducted during the induction and development ofcolon tumors. The effect of selenium supplementation before,during, and after DMH treatment on the parameters assayedhas been tabulated at 10-week intervals or other selected time

periods for brevity. One aim of this study was to obtain diagnostic and therapeutic markers of colon cancer. Such markersimplicated by the data presented include increased WBC,reduced hemoglobin, increased SGOT and alkaline phosphatase, and perhaps decreased acid phosphatase. While eachindividual marker is not specific for colon cancer, the collectivechanges in all of these parameters appear to correlate with

NOVEMBER 1981 4463



M. M. Jacobs et al.

DMH treatment, with colon tumor development, and with theeffect of selenium on DMH-induced carcinogenesis.

A second aim of this study was to suggest a mechanism bywhich selenium inhibits DMH-induced neoplasia. Consideration

is first given to the GSHP data reported, then to an interpretation of the /?-glucuronidase data, and finally to plausible mech

anisms of selenium inhibition.Our initial intent in measuring GSHP activity was to evaluate

the selenium status of the animals, anticipating that selenium-supplemented rats would have increased selenium-dependent

enzyme activity. On the contrary, according to Table 5, theactivity of both forms of the enzyme was lowest in the seleniumonly group, intermediate in the selenium- and DMH-treatedgroups, and highest in the untreated control. The selenium-

dependent GSHP efficiently uses H2O2 as substrate, while theselenium-independent GSHP more efficiently uses eumene hy-

droperoxide as substrate (2, 23). The relative contributions ofeach enzyme to the total activity measured with each substrateis not known for the samples we assayed. Using H2O2, thegreater range in activity was observed. Other inhibitors ofcarcinogen-induced neoplasia increase the glutathione S-

transferase activity (3). Implicit to this observation is an increase in glutathione. Perhaps selenium induces an abundanceof glutathione, and this in turn induces substrate inhibitionyielding a decrease in GSHP activity in the selenium only-

supplemented group. Alternatively, the glutathione substratemay be used more efficiently by the transferase having decreased substrate availability for the GSHP. In a report by Scottet al. (30), the supplementation of the diets of rats with increasing levels of selenium caused corresponding increases in theGSHP activity up to a point. At the highest level of selenium (5ppm), the enzyme activity decreased. Perhaps a similar relationship is being observed in this study.

Epidemiological studies indicate that Americans consuminga mixed "Western" diet had higher levels of fecal bacterial ÃŸ-

glucuronidase than American vegetarians, Seventh Day Adven-

tists, Chinese, or Japanese (28). Presumably a high beef dietis a risk factor altering bacterial enzymes responsible for theconversion of procarcinogens to proximal carcinogens. To testthis experimentally, rats treated with DMH were administereda beef diet simulating a "Western" diet. A higher colon tumor

incidence resulted. Supplementation of the beef diet with Lac-

tobacillus acidophilus reduced the colon tumor incidence andthe fecal bacterial ÃŸ-glucuronidase (10). Since oral seleniumreduced the colon tumor incidence in our studies, we assayedthe bacterial /S-glucuronidase to determine whether seleniumreduced this enzyme activity as a suggested mechanism ofaction. We observed that the bacterial /?-glucuronidase washigher in DMH-treated rats than untreated controls but wasgreatest in selenium- and DMH-treated animals. Hence, selenium inhibition of DMH-induced colon tumors could not beattributed to this enzyme. The increase in /?-glucuronidase in

the selenium only control perhaps reflects a response to otheras yet unidentified glycoconjugates also recognized as suitablesubstrates by the hydrolytic enzyme. These substrates may benormal constituents of the mucous secretions in the colon butare perhaps in greater abundance at 41 weeks of age followingprolonged exposure to the subtoxic selenium supplement.

Current data suggest that DMH is activated through thefollowing sequence: DMH -Â» azomethane -Â» azoxymethaneâ€”¿�Â»MAM. Under physiological conditions, MAM is unstable and

breaks down to form the strong alkylating agent, methyldiazon-

ium hydroxide, and formaldehyde (6, 8). Laquer et al. (22)found the MAM glycoside (cycasin) to be a potent carcinogenin conventional rats but not in germ-free rats (22). From multiple

bioassays of synthetic and natural metabolites, Laquer (20, 21 )reported that the aglycone (MAM) produced the same effectsin germ-free rats that the intact glucoside produced in conven

tional rats. Their data indicated that the intestinal bacteriapossessed the ÃŸ-glucosidase for liberation of the aglycone

from the conjugate. Studies with inhibitors have increased ourknowledge of the activation sequence for carcinogens as wellas the mechanism and loci of inhibitor actions (36, 38). Forexample, disulfiram and related compounds that contain acarbon disulfide moiety inhibit DMH-induced neoplasia. Thetargets of disulfiram appear to be inhibition of the A/-oxidation

of azomethane and the hydroxylation of azoxymethane to MAM(7, 37). Pyrazole acts at other loci, the azoxymethane hydrox-

ylase as well as the alcohol dehydrogenase activation of MAM(40). Although specific metabolites and enzymes indicatedabove were not assayed in the present study, selenium couldsimilarly act at one or more of these loci causing inhibition ofDMH-induced as well as MAM-induced neoplasia reported

elsewhere (14).

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1981;41:4458-4465. Cancer Res Maryce M. Jacobs, Curtis F. Forst and Faith A. Beams Dimethylhydrazine-induced Colon Cancer in RatsBiochemical and Clinical Effects of Selenium on

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