Indian 10urnal of Experimental Biology Vol. 37, January 1999, pp. 50-55

Absence of in vivo mutagenic potency of heated and fried oils in mice

K Narasimhamurthy, Muralidhara & P L Raina

Department of Biochemistry and Nutrition, Central Food Technological Research Inst itute, Mysore 570 013, India

Received 22 January 1998; revised 18 August 1998

The mutagenic potency of heated and fried oils(under laboratory conditions), viz. peanut, sesame and coconut oils were studied employing two well validated in vivo marmnalian assays, viz. bone marrow micronucleus assay and the sperm morphology assay in albino ' mice. In the bone marrow micronucleus assay oral doses of fresh, heated and fried oils (7.5 ml and 30.0 mJ/kg body weight) did not induce any increase in the incidence of micronucleated polychromatic erythrocytes. [ntragastric administration of oils at the highest dose (30.0 mJ/kg body weight/day) for 5 consecutive days in male mice did not produce any morphological effect either on the testicular or epididymal we ights and histology. No significant alterations were observed in the caudal sperm counts at any of the sampling intervals and also there was no treatment related increase in the incidence of sperm head abnormalities. It is concluded that controlled thermal treatment as well frying of these oils did not produce appre~iable amount of mutagens which could be detected under in vivo situations.

In India deep fat fr ied products form the major route through which oils and fats are consumed. Heating and frying changes the composition of oil s and fa ts and during the process some new compounds are formed. When oils and fats are subjected to heat for prolonged periods of time as noticed In commercial practices there is tendency for exessive accumulation of oxidised, polymeric and cyclic compounds. The amount of decomposition products formed increase gradually the longer the oils are heated l

. Such unavoidable chemical reactions cause formation of both volatile and nonvolatile decomposition products that may affect human hea lth2

.3

Available literature on the possible mutagenic activity of heated/fried oi ls is both scanty and conflicting. Majority of the researchers have employed bacterial systems (Ames assay) to evaluate the mutagenic activity of oils per se or condensation of volati les formed during heating/frying or extracts of vanous fried products4

•7 Few researchers have

attributed posItIve mutagenic effects to heterocyclic amInes and other pyrolysates formed in foods during deep fat frying which in

small amounts may possibly migrate into the frying fat als08

.9

. Method of food preparation has a significant influence on the formation of byproducts which may possess considerable mutagemc actIvIty. Heterocyclic amines formed during cooking of meat and many of the amino acids and sugars present in foods are found to be precursors in the formation of mutagenic compounds during frying . Data on the in vivo mutagenic potency of various widely used heated/fried oils is limited 10-13

In view of the above, the present study aimed at studying the possible mutagenic potency of heated/fried oils, vi z. peanut oil, sesame ' oil and coconut oil WhlCh constitutes nearly 60% of total availability in India l 4 under laboratory conditions of heating and frying. The bone marrow micronucleus assay is a very efficient and sensitive in vivo test system to detect cytogenetically active compounds as developed by Schmid l5 and the sperrn morphology assay as proposed by Wyrobeck and Bruce l6 for the rapid detection of potentia l reproductive toxins, mutagens and carcinogens were employed in the study.

NARASIMHAMURTHY et al. : ABSENCE OF IN VIVO MUTAGENIC POTENCY OF HEATED OILS IN MICE 51

Materials and Methods Heated (HO) and Fried oils (FRO)-Peanut oi l

(PNO), sesame oil (SO) and coconut oil (CNO) of edible grade were purchased from the local market. The oils were heated in stainless steel containers in an hot air oven at 180o±2°C . continuously for 72 hr. The oils were periodically stirred once every 4 hr. After the completion of heating the oils were removed from the oven, cooled to room temperature and transferred into glass containers, sealed under nitrogen and stored at 4°C until further use .

Freshly peeled potato slices were fried in an iron vessel in batches in each of the above oil continuously for two and half hours. No new fresh oil was added during fry ing to compensate for the loss due to uptake of oil by the frying materiaL After the completion of frying the oils were cooled to room temperature, transferred to glass containers, sealed under nitrogen and stored at 4°C until further use.

Animals and diet- Male Swiss albino mice of eFT strain (30-35g) bred and reared in the animal house facility at our animal house facility, were held in plastic cages in groups . During a week of acclimatization and throughout the experimental period, they were maintained on mouse pellets (Gold Mohur, Lipton India Ltd.) ad libitum and had free access to water.

Bone marrow micronucleus assay--6-week-old male mIce (30-35g) were intragastri~ally

admini stered either fresh (unheated) (FO), heated (HO) or fried oil (FRO) at doses of 7.5 ml/kg body weight (low dose) and 30.0 ml/kg body weight (high dose) in split dose at 0 and 24 hr. These doses were computed based on our earlier dietary studies in rats 17 . (The high dose was the maximum practical dosage based on the stomach volume of the mouse and was considered as physiologically acceptable). The animals were killed by cervical dislocation at 30 hr and the bone marrow preparations were made using human AB serum as described previously'8. The femoral cells were flushed out with human AB serum and the cellular suspension was centrifuged at 2000 rpm for 5 min.

Smears were preppred from the resulting pellet following the procedure of Schmid IS. Four mice were used for each dose and four slides were

prepared for each animal and the incidence of micronuclei (MN) in an average of 4000 polychromatic erythrocytes (PCE) and corresponding normochromatic erythrocytes (NCE) were scored for each animaL Mice administered an acute dose of cyclophosphamide (20 mg/kg body weight, ip) served as the positive controL The MN data was analysed by student's- t-test.

Subacute study: Caudal sperm counts and sperm morphology assay--Groups of male mice (8-10 week old) were administered (oral) with each of the above oil either fresh, heated or fried at the highest dose (30.0 ml/kg body weight) twice a day for five consecutive days. The animals were killed by cervical dislocation at 1, 3, 5 and 7 week after the end of each treatment. After recording the fresh weights of the epididymes and testes , a portion of the tissue was fixed in freshly prepared Bouin's fluid and pro'cessed for histological examination. Fresh cauda held in 0.9% NaCl were prQcessed according to the procedure of Muralidhara and Narasimhamurthy ' 9 to determine the sperm count. Briefly,aliquots of sperm suspension were stained with 1 % Eosin Y and the sperms were scored for abnormal sperms adopting the procedure · of Wyrobeck and Bruce '6. A minimum of 1000 sperms were analysed per animaL While the data on body weight, epididymal and testicular weights were analysed by Student's t test the data on sperm counts and sperm abnormalities were analysed by the Mann-Whitney U test.

Results

Bone marrow micronucleus assay--Data on the incidence of MN in peE and NeE are shown in Table 1. Oral administration of heated/fried oil did not produce any significant increase in the incidence of MN in peE's of bone marrow. The average spontaneous incidence of MN in peE and NCE in various unheated (fresh) oi l controls ranged from 0.25 to 0.3 1% and 0.14 to 0.19% respectively. The incidence of MN in mice treated either HO or FRO even at the highest dose was highly comparable to those of FO administered controls. The incidence of micronucleated peE among the control mice was in the same range as reported previously by us l 8.1O

.

52 INDIAN J. EXP. BlOL. , JAN UARY 1999

Table I- Incidence of micronuclei (MN) in bone marrow erythrocytes of mice administered (oral) fresh (FO), heated (HO) and fried (FRO) oils

Dosage Group PCE with MN (%) NCE with MN (%) PCE (%) PIN ratio (ml /kg

bodywt)

Peanut oil

FO 0.31 O±O.O 14 0.145±0.021 51.2 1.05 (49/16000) (221l 5220)

7.5 HO 0.287±O.026 0.163±0.011 52.8 1.1 2 (46/1 6000) (251143 10)

FRO 0.262±0.027 0.140±0.012 52.9 1.25 (42/ 16000) (20112780)

FO 0.300±0.0 16 0.165±0.014 51.4 1.06 (48/16000) (25/15110)

30.0 HO 0.325±0.0 18 0.195±0.0 17 55.6 1.25 (52/1 6000) (20/12780)

FRO 0.313±0.022 0.177±0.009 53 .1 1.\3 (50/16000) (25/141 10)

Sesame oil

FO 0.281±0.024 0. 148±0.006 51.8 1.08 (45/16000) (22/ 14880)

7.5 HO 0.306±0.032 0.205±0.0 12 55.0 1.22 (49/1 6000) (27113 100)

FRO 0.294±0.020 0.2 12±0.0 11 53.0 1.13 (47/16000) (30/14180)

FO 0.250±0.0 18 0.1 80±0.020 53.5 1.15 (40/16000) (25/13900)

30.0 HO 0.350±0.021 0.214±0.022 53 .3 1.14 (56/1 6000) (30/14000)

FRO 0.310±0.017 0.20 1±0.029 53 .5 1.15 (49/16000) (28/1 3900)

CQconut oil

FO 0.318±0.054 0. 179±0.0 II 52.4 1. 15 ( J 5/ 16000) (26/14540)

7.5 HO 0.325 ±0.022 0. 194±0,0 19 53 .5 1.1 5 (52/ 16000) (27113915)

FRO 0.300±0.0 16 0.2 12±0.024 53 . 1 1.13 (48/16000) (30/141 12)

FO 0.250±0.020 0. 197±0.024 54.9 1.22 (40i16000) (26/1 3 160)

30.0 HO O.268±O.026 0. 192±0.026 55.1 1.22 (43/16000) (25 / 13050)

FRO 0.288±0.032 0.170±0.008 53.1 1.13 (46/ 16000) (24/14 150)

CP, 20.0 2.250±0.1 66· 0.276±0.178* 37.1 * 0.59· mg/kg bw (360/1 6000) (75/27155)

Values are mean ± SE of 4 animal s per dose; values in parentheses are MN/total cells scored. • Values are significantly different from others in column (P<O.OO I, Student's 't' test) CP--Cyclophosphamide

NARASIMHAMURTHY et at.: ABSENCE OF IN VIVO MUTAGENIC POTENCY OF HEATED OILS IN MICE 53

Furthermore no decrease in the PCEINCE ratio was observed in any of the animals treated with any of the heated/fried oils. In contrast, the positive mutagen cyclophosphamide (CP) induced nearly a 7-fold increase in the frequency of micronucleated PCE and a 3-fold increase in the micronuc1eated NCE. In addition, CP produced a significant decrease in the PIN ratio suggesting a marked ' cytotoxic effect, and these positive responses clearly indicate the sensitivity of the CFT strain.

heated/fried oil treated mice assayed at different intervals of 1, 3, 5 and 7 week post-treatment showed no significant alteration compared with unheated oils (Table 2). . Furthermore, there were no treatment related changes in the incidence of sperm head abnonnalities, and the percentage of abnormal sperms in treated mice were on par with those of control unheated oils. The sperms analysed at 1, 3, 5 and 7 week post­treatment presumably correspond with the treatment of mature sperms, spermatids,

of spermatocytes and spermatozoa respectively. Subacute treatment--Oral administration heated/fried oils on 5 consecutive days at a dose of 30 ml/kg body weight did not induce any clinical signs of toxicity or mortality in mice. Further, analysis of data on testicular and epididymal weights among the control and various treatment groups showed no significant differences (Data not shown). Microscopic examination of testis and epididymis revealed no significant alterations in the histoarchitecture in

Discussion

any of the groups. Epididymal sperm counts and sperm

There has been considerable concern with regard to the formation of mutagenic/carcinogenic compounds upon heating and frying of fats. Interestingly, the available findings are rather equivocal and majority of the studies have employed bacterial systems lO

. 2 1-23. Studies on the in vivo mutagenic potency of heated/fried oils is scanty. In this context, the present study aimed at assessing the possible mutagenic effects of selected heated and fried oils employing short term in vivo mutagenicity assays. morphology assay--The caudal sperm counts of

Table 2-Epididymal spenn counts and incidence of abnonnal spenns in mice admin istered (oral) heated, fried and fresh peanut (PNO), sesame (SO) and coconut oil (CNO) on five consecutive days (dose-30.0 mllkg .body wt)

Week Parameter PNO SO CNO I Spenn Fa HO FRO Fa HO FRO Fa HO FRO

count*

214.0 222 .5 198.0 218 .8 208 .3 226.0 225 .5 2 15 .8 211.2 ± 17.8 ± 23 .21 ± 113 .3 ± 17.4 ± 13. 10 ± 11.61 ± 11.61 ± 15.2 ± 11.75

Abnonnal 1.67 1.85 1.95 1.56 2.01 1.73 1. 52 1.42 170 sperms (%) ± 0.3 8 ± 0.22 ± 0.60 ± 0.36 ± 0.35 ± 0.30 ± 0.30 ± 0.13 ± 0.11

III Sperm 196.3 188.0 209.3 213 .3 1973 227.2 226.0 215 .2 21 8.8 count ± 8.5 ± 4. 11 ± 6.47 ± 10.72 ± 13.3 ± 15 .67 ± 6.61 ± 11.1 0 ± 4 .20 Abnormal 1.59 1.65 1.66 1.77 2.08 1.60 I. 7 1 1.02 1.52 sperms (%) ± 0.30 ± 0.3 ± 0.15 ± 0.53 ± 0.51 ± 0.30 0.15 ± 0 03 ± 0.18

V Sperm 2 13.0 2.3 .3 206.3 220.0 21 8.0 21 0.5 205 .3 195.40 r' count ± 6.2 ± 20.5 1 ± 11.S3 ± 15.27 ± 20.4 ± 13.2 ± 21.3 ± 22 .50 6.03 Abnormal 1.72 1.52 1.73 1.95 1. 8 1 1.59 1.48 1.68 1.42 sperms (%) ± 0.34 ± 0.18 ± 0.26 ± 0.46 ± 0. 51 ± 0.25 ± 0.5 1 ± 016 0.24

VII Sperm 207.0 194.4 218.0 222.0 2 12.3 2 19.0 227 .3 225.4 221 .2 count ± 1.90 ± 23.6 ± 13 .28 ± 8.19 ± 32.6 ± 6.64 ± 28.9 ± 24.84 ± 7.60 Abnormal 1. 77 1.73 1.65 1. 82 1.90 1. 55 1.66 1.44 1. 72 sperms (%) ± 0.25 ± 0.26 ± 0.25 ± 0.3 1 ± 0.42 ± 0.3 1 ± 0.24 ± 0.08 ± 0.17

FO-Fresh o il ; HO-Heated oil ; FRO-Fried oil *Values shou ld be multi plied by 105 to get the actual sperm counts per cauda epididymi s Values are mean ± SE of 6 animals each. No sign ificant di ffe rence between treatment and control oi l groups (Mann-Whitney U test)

54 IN DIAN 1. EXP. BIOL. , JANUARY 1999

The results obtained in the bone marrow micronucleus assay clearly suggested the absence of any substances with clastogenic. potency either in heated or fried oils. Although we do not rule out the qualitative presence of any mutagenically active compounds in heated/fried oils, they may not be present in appreciable levels. The suitability of our strain of mice to evaluate the in vivo mutagenic potency has been established earlierIS.20. Accordingly the . incidence of micronucleated PCE among fresh, heated or fried oils were highly comparable to those of historical controls24.IS.20 . Furthermore, the normal PIN ratio among the treated mice suggests absence of any cytotoxic effect on the proliferation of bone marrow cells.

Similar reports on the absence of mutagenic activity in bacterial system has been documented earliers.27

. Lack of any possible mutagenic response in the in vivo short term test could be attributed to (a) low concentra"tion of clastogenetically active compounds (b) no clastogenic substances in the oils due to controlled, heating or fryi ng and (c) inherent characteristics of the oil employed.

The negative response could be further explained on the basis of the presence of various anti oxidative compounds like butylated hydroxy anisole (BHA) and butylated hydroxy toluene (BHT) in the oils27. II. Sesame oil employed in the study is known to contain appreciable levels of antioxidant phenolic compounds such as sesamol and sesamin2s which are known to possess antioxidant and antimutagenic properties29.3o

. Furthermore, this may be the probable reason why foods fried ii1 sesame oil (e.g. potato chips) stay for longer periods than when fried in other oi ls3 1

• Coconut oil being a highly saturated oil (with more than 90% of saturated fatty acids) offers a strong resistance to oxidative rancidity and the oxidation, polymerisation and generation of free radicals are thus minimised32. In general, the absence of mutagenic response may be related to the low amount of peroxide value In the heated/fried oils lo . 17.

Similar negative response was evident in the germ cells also. Both heated/fried oils tested at the highest practical dose failed to alter any of the

parameters studied viz. testicular, epididymal weights and caudal sperm counts suggesting lack of any deleterious effect. Further, these oils did not induce any increase in the percentage of sperm head abnormalities suggesting non-interference with normal sperm production and development.

In conclusion, the present study showed that heating of oils at 180°C for 72 hr. or frying (under laboratory conditions) did not generate any significant mutagenic load as the oils evaluated failed to induce a positive mutagenic response in short term mutagenicity assays.

Acknowledgement

The authors wish to thank Dr. S G Bhat, Head, Department of Biochemistry and Nutri tion and Dr V Prakash, Director of the Institute for their encouragement and support.

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