Effect of cypermethrin, carbendazim and their combination on male albino rat serum

Abstract

Alpha-cypermethrin and carbendazim are synthetic; α-cypermethrin belongs to a class of synthetic pyrethroids and carbendazim belongs to the class of carbamate fungicides. The current study was carried out to evaluate the low-dose exposure of individual and mixed forms of cypermethrin and carbendazim. α-cypermethrin was used at 0.06, 0.12, 0.30 and 0.60 mg/kg body weight (bw), carbendazim was at 0.48, 0.96, 2.4 and 4.8 mg/kg bw and combined doses (cypermethrin: 0.06, 0.12, 0.30 and 0.60 mg/kg.bwt + carbendazim: 0.48, 0.96, 2.4 and 4.8 mg/kg.bwt) for 12 h and 24 h. The biochemical parameters and serum markey enzymes were analysed. The biochemical parameters include serum total protein, glucose, cholesterol, urea, uric acid, calcium, phosphorous, albumin and creatinine and serum marker enzymes such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT) and amylase were ascertained. Results indicated simultaneous changes in serum marker enzyme activity (ALT, AST, ALP, GGT and amylase) and biochemical markers (*P < 0.05, **P < 0.01 and ***P < 0.001). The experimental results indicate that even low-dose use of the synthetic pyrethroid carbamate and their combined form results in consequential negative effects on cell function.

Cypermethrin belongs to a class of insecticides known as synthetic pyrethroids. Synthetic pyrethroids are man-made insecticides created to mimic the chemical properties of the naturally occurring insecticide pyrethrum, which comes from the crushed petals of the Chrysanthemum flower. Synthetic pyrethroids, like cypermethrin, are often preferred to the natural forms as active ingredients because they offer the added bonus of remaining effective for longer periods of time (Ellenhorn et al. 1977). The introduction of novel, more toxic and rapidly disseminating pesticides into the environment has necessitated an accurate identification of their potential hazards to human health. Although these toxic chemicals have become an integral part of the ecosystem, many of them remain extremely toxic to mammals and other non-target creatures. Pyrethroid-related sensory irritation in the respiratory tract was studied (Pauluhn 1996) by exposure studies in mice and rats. The guinea pigs received 0.1 ml of a 0.01, 0.1 or 1.0% solution of cypermethrin in ethanol or a 1, 10 or 20% solution of cypermethrin (w/v) in corn oil on the skin. Sensory stimulation was quantified by counting the number of times each animal turned to lick or bite its treated flank in preference to the untreated flank. Skin stimulation was observed during a 2-h period at all dose levels except the lowest (Dewar 1971).

Carbendazim is a systemic broad-spectrum fungicide, controlling a wide range of pathogens. It is also used as a preservative in paint, textile, paper-making and leather industry, as well as a preservative of fruits. Carbendazim exposure was associated with impaired liver function, altered enzyme activity and changes in, haematopoiesis and reproduction in various mammals (Dreisbach 1983). Cypermethrin and carbendazim are used widely to prevent and control insects and pests. In our previous study, both cypermethrin and carbendazim independently produced differential effects in male albino rats at single fixed dose level. Serum marker enzymes such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT) and amylase and biochemical markers such as serum total protein, glucose, cholesterol, albumin, urea, uric acid, calcium, creatinine and phosphorous are representative of the functional status of homoeostaticsis. Thus a in the serum content of these marker enzymes and biochemical parameters reflects the overall health status of animals when they have been subjected to exogenous modulants such as pesticides. Hence, the present study was designed to evaluate time- and dose-dependent effect of cypermethrin, carbendazim and their combinations using male albino rat serum as a reporter system.

Materials and Methods

Materials

Alpha-cypermethrin (95.6% pure) and carbendazim (98.3% pure) were purchased from Gharda Chemicals Ltd., Mumbai, India. All chemicals and laboratory wares were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Glass distilled water was used for the preparation of all reagents. Male Wistar strain-albino rats weighing 180–200 g were used for the investigation. High-speed centrifuge (Sorvall RC-5C Plus Superspeed Centrifuge (SRC5C), GMI, USA.) and UV-Visible Spectrophotometer (Shimadzu-UV-1800, Nishinokyo-Kuwabara-cho, Nakagyo-ku, Kyoto, Japan) were also used for this investigation. All the procedure dealing treatment and sacrifice of rats was carried out according to internationally ethical committee regulations. The animals were housed under controlled temperature and hygiene conditions with 12 h of light and dark cycle throughout the experimental period. Commercial rat chow with free access to drinking water ad libitum was provided for the animals.

Methods

The experimental approach was aimed to investigate the effect of the insecticide α-cypermethrin and the fungicide carbendazim on the biochemistry of blood and serum in male albino Wistar strain rats. Control and treated group contained six animals each. Dose level of cypermethrin was used at 0.06, 0.12, 0.30 and 0.60 mg/kg bw. Carbendazim was at 0.48, 0.96, 2.4 and 4.8 mg/kg bw and their equal combination (cypermethrin: 0.06, 0.12, 0.30 and 0.60 mg/kg bw + carbendazim: 0.48, 0.96, 2.4 and 4.8 mg/kg bw) for durations of 12 and 24 h in the different groups of rats. The compounds were instantly dissolved in 0.1 ml ethanol and were administered intradermally. For the determination of acute effects usually the compounds are administered via the intra dermal route because the doses administered can spread rapidly all over the body and produce the full effect of the dose that has been administered, whereas administration via a dermal route results in slower spreading and may produce major local effects. The time intervals selected for investigation were 12 and 24 h following the administration of each compound in all the studies. Blood was collected by cardiac puncture into a tube containing an anti-coagulant (1.15% EDTA), and serum was prepared without the use of the anti-coagulant.

Biochemical parameters

Protein content was estimated by the method described by Muthuraman and Srikumar (2010). Albumin content was measured by the method described by Nwanjo et al. (2007). Total cholesterol was estimated by the method described by Gohil et al. (2011), and blood glucose using the method described by Sani et al. (2009). Urea content was measured by Using the method described by Saxena and Saxena (2010). Uric acid content was estimated by the method of Caraway (1963). Creatinine content was measured by the method described by Peake and Whiting (2006). Calcium content was measured by using the method described by Gitelman (1967). Phosphorus content was measured by using the method described by Goldenberg (1966).

Enzyme activities

Alanine transaminase, AST, ALP and GGT enzyme activities were measured by standard methods (Muthuviveganandavel et al. 2008). Amylase activity was measured using the method described by Proctor et al. (1991).

Statistical analysis

All the values were expressed as mean ± SEM. Statistical analysis was done using SPSS 11 (Statistical Package). The statistical significance of differences between the means was assessed by anova followed by Dunnett test. P values < 0.05 were considered as significant.

Results

This study used cypermethrin and carbendazim, two different pesticide compounds at lower doses individually and in combination to determine their metabolic impact in male albino Wistar strain rat serum.

Serum total protein content did not exhibit the quantum of variation at different times studied that has been as noted for the tissue total protein content (8.3%). A maximum variability of <10% below control was noted with 2.4 mg/kg bw of carbendazim (Table 1, *P < 0.05, *P < 0.01). Owing to the differences in the control level of serum albumin between 12 and 24 h, change in the albumin content (0.14–0.20 and 0.14 g/dl) at 24 h because of the use of different concentrations of cypermethrin because of 0.48 mg/kg.bwt of carbendazim and because of the different concentrations of the combination considered (Table 1).

Table 1.

Rat serum total protein and albumin contents in control and treated samples. Cypermethrin, carbendazim and cypermethrin + carbendazim (each) at 12- and 24-h duration

	Total Protein (g/dl)		Albumin (g/dl)
Groups	12 h	24 h	12 h	24 h
CONTROL	6.1 ± 0.16	6.21 ± 0.09	3.2 ± 0.17	3.5 ± 0.22
CYP-0.06 mg/kg bw	5.7 ± 0.28	6.1 ± 0.19	3.8 ± 0.19a,c	4.1 ± 0.22a,c
CYP-0.12 mg/kg bw	5.66 ± 0.24	6.13 ± 0.24	3.6 ± 0.25	4 ± 0.15
CYP-0.30 mg/kg bw	5.5 ± 0.17a,c	6 ± 0.39	3.45 ± 0.18	4.2 ± 0.24a,c
CYP-0.60 mg/kg bw	5.5 ± 0.21a,c	6.1 ± 0.12	3.4 ± 0.2	4.1 ± 0.17a,c
CAR-0.48 mg/kg bw	5.5 ± 0.21a,c	6 ± 0.09	3.3 ± 0.18	4 ± 0.2
CAR-0.96 mg/kg bw	5.25 ± 0.17b,d	5.9 ± 0.08	3.2 ± 0.2	3.2 ± 0.17
CAR-2.4 mg/kg bw	5.1 ± 0.25a,d	5.7 ± 0.08a,d	2.9 ± 0.27a	2.9 ± 0.2a,c
CAR-4.8 mg/kg bw	5.2 ± 0.19a,d	5.6 ± 0.11a,d	3.2 ± 0.2	3.1 ± 0.27
CYP-0.06 + CAR-0.48 mg/kg bw	5.8 ± 0.22	6 ± 0.07	2.8 ± 0.21b	3.8 ± 0.25
CYP-0.12 + CAR-0.96 mg/kg bw	5.7 ± 0.24	5.9 ± 0.11	3.2 ± 0.19	4 ± 0.32a
CYP-0.30 + CAR-2.4mg/kg bw	5.8 ± 0.22	6.1 ± 0.08	3.3 ± 0.24	3.5 ± 0.19
CYP-0.60 + CAR-4.8mg/kg bw	5.1 ± 0.2a,d	6 ± 0.12	3.5 ± 0.29	3.8 ± 0.26

Values are mean ± SEM from 6 rats in each group.

^aP < 0.05,

^bP < 0.01,

^eP < 0.001: Compared between the control and treated groups.

^cP < 0.05 and

^dP < 0.01: Compared between the treated groups. Data analysed by one-way anova followed by Dunnett test.

Serum urea levels remained around the 42–50 mg of control, and exhibited an increase attributed to cypermethrin and carbendazim independently only at 12 h. Surprisingly the control urea level registered below 30 mg/dl by 24 h along with the treated samples (Table 2). Changes in blood uric acid content (18 and 32 mg/dl) werenoted due to carbendazim alone at 12 and 24 h, whereas higher levels of the combination yielded perceptible increase over the control at 12 h (Table 2).

Table 2.

Rat serum urea and uric acid contents in control and treated samples. Cypermethrin, carbendazim and cypermethrin + carbendazim (each) at 12- and 24-h duration

	Urea (mg/dl)		Uric acid (mg/dl)
Groups	12 h	24 h	12 h	24 h
CONTROL	48 ± 3.9	28 ± 2.7	12.7 ± 0.19	11 ± 0.16
CYP -0.06 mg/kg bw	47.5 ± 4	19 ± 1.4a,d	9 ± 0.12a,d	9.8 ± 0.19a,d
CYP-0.12 mg/kg bw	42 ± 2.3	20 ± 1.6d	9.8 ± 0.2a,d	9.9 ± 0.12a,d
CYP-0.30 mg/kg bw	39 ± 3.3a,d	25 ± 2.2	10.1 ± 0.22a,d	10.1 ± 0.17a,d
CYP-0.60 mg/kg bw	40 ± 2.6a,c	26 ± 1.7	10.4 ± 0.16a,d	10.5 ± 0.26a
CAR-0.48 mg/kg bw	46.5 ± 4	25 ± 1.8	13.1 ± 0.14	13.6 ± 0.19b,d
CAR-0.96 mg/kg bw	38 ± 2.5a,d	15 ± 1.1b,d	14 ± 0.21d	14 ± 0.43b,d
CAR-2.4 mg/kg bw	43 ± 2.6	20 ± 1.0d	14.4 ± 0.26a,d	14.3 ± 0.33b,d
CAR-4.8 mg/kg bw	46 ± 4.1	16 ± 0.9b,d	15 ± 0.28a,d	14.5 ± 0.35b,d
CYP-0.06 + CAR-0.48 mg/kg bw	48 ± 2	26 ± 1.7	12 ± 0.28d	10.9 ± 0.3
CYP-0.12 + CAR-0.96 mg/kg bw	47 ± 2	29.5 ± 2.3	13 ± 0.21	11.2 ± 0.2
CYP-0.30 + CAR-2.4mg/kg bw	48 ± 1.8	29.5 ± 1.3	13.1 ± 0.22	11.8 ± 0.22d
CYP-0.60 + CAR-4.8mg/kg bw	34 ± 2.5b,d	25 ± 1.9	13.7 ± 0.22d	12 ± 0.21d

Values are mean ± SEM from 6 rats in each group.

^aP < 0.05,

^bP < 0.01 and

^eP < 0.001: Compared between the control and treated groups.

^cP < 0.05 and

^dP < 0.01: Compared between the treated groups. Data analysed by one-way anova followed by Dunnett test.

Serum glucose content was increased (36 & 60, 39 & 112 mg/dl) with 0.06 mg/kg bw and 0.60 mg/kg bw of cypermethrin, 0.48 mg/kg bw and 4.8 mg/kg bw of carbendazim respectively. Serum glucose content also increased (19 & 42, 88 & 43 mg/dl) with the combination of 0.30 mg/kg bw and 0.60 mg/kg bw of cypermethrin, 2.4 mg/kg bw and 4.8 mg/kg bw of carbendazim at 12 h. The increased serum glucose content noted at 24h, remained above the 24-h control; significant differences (125 mg/dl) were seen with the 2.4 mg/kg.bwt carbendazim dose (Table 3). Serum cholesterol level remained more or less stationary at 12 h with the carbendazim and with the combination. Cypermethrin alone increased serum cholesterol above 20 mg/dl at 12 h. However, the cypermethrin effect was over shadowed by increasing doses of carbendazim at 24h time point, registering 60% increase in cholesterol content. Combination of cypermethrin and carbendazim, however, brought back the serum cholesterol level equivalent to that of control at 24 h (Table 3).

Table 3.

Rat serum glucose and cholesterol contents in control and treated samples. Cypermethrin, carbendazim and cypermethrin + carbendazim (each) at 12- and 24-h duration

	Glucose (mg/dl)		Cholesterol (mg/dl)
Groups	12 h	24 h	12 h	4 h
CONTROL	130 ± 8.2	60 ± 3.8	45 ± 2.6	43 ± 1.3
CYP-0.06 mg/kg bw	141 ± 8	72 ± 4a	59 ± 2a,d	45 ± 1.1
CYP-0.12 mg/kg bw	137 ± 6	69 ± 4	52 ± 0.8d	48 ± 1.7c
CYP-0.30 mg/kg bw	155 ± 7a,d	66 ± 5	47 ± 1.6	55 ± 1.4a,d
CYP-0.60 mg/kg bw	184 ± 6b,d	62 ± 3	48 ± 2	59 ± 1.4a,d
CAR-0.48 mg/kg bw	109 ± 6b,d	95 ± 6a	39 ± 1.7d	62 ± 2.6a
CAR-0.96 mg/kg bw	119 ± 5a	98 ± 7a,d	43 ± 2.3	74 ± 2.2b,d
CAR-2.4 mg/kg bw	122 ± 4	135 ± 6a,d	48 ± 1.9	75 ± 3b,d
CAR-4.8 mg/kg bw	245 ± 9b,d	86.8 ± 6a,d	49 ± 1.6	77 ± 2.1b,d
CYP-0.06 + CAR-0.48 mg/kg bw	186 ± 6b,d	67 ± 4	47 ± 1.4	42 ± 1.3
CYP-0.12 + CAR-0.96 mg/kg bw	93 ± 5b,d	62 ± 5	42 ± 1.6	46 ± 2.2
CYP-0.30 + CAR-2.4mg/kg bw	98 ± 5a,d	74 ± 5a,c	44 ± 1.7	46 ± 1.9
CYP-0.60 + CAR-4.8mg/kg bw	51 ± 7b,d	68 ± 5	50.33 ± 2.2d	47 ± 1.4

Values are mean ± SEM from 6 rats in each group.

^aP < 0.05,

^bP < 0.01 and

^eP < 0.001: Compared between the control and treated groups.

^cP < 0.05 and

^dP < 0.01: Compared between the treated groups. Data analysed by one-way anova followed by Dunnett test.

Control serum creatinine level ranged between 0.3 and 0.5 mg/dl. Increase in serum creatinine content (50, 20 & 40 and 20 mg/dl) was therefore noted only with 0.60 + 4.8 mg/kg bw combination at 12 h, 2.4 mg/kg bw and 4.8 mg/kg bw carbendazim at 24 h and 0.60 + 4.8 mg/kg bw combination at 24 h (Table 4). Significant changes in serum calcium were noted in -nearly all treatments (Table 4). Serum phosphorous content, showed increase, following cypermethrin and carbendazim treatment. At 12 and 24 h (8 and 26 mg/dl) following 0.48 mg/kg bw and 4.8 mg/kg bw carbendazim (Table 5), serum amylase showed no appreciable change. Amylase enzyme activity significantly increased at 0.96, 2.4 mg/kg bw and reduced at 4.8 mg/kg bw of carbendazim, following 24 h of administration. The combination of cypermethrin and carbendazim also increased amylase enzyme activity (0.12 + 0.96 and 0.60 + 4.8 mg/kg bw, Table 5).

Table 4.

Rat serum creatinine and calcium contents in control and treated samples. Cypermethrin, carbendazim and cypermethrin + carbendazim (each) at 12- and 24-h duration

	Creatinine (mg/dl)		Calcium (mg/dl)
Groups	12 h	24 h	12 h	24 h
CONTROL	0.4 ± 0.04	0.5 ± 0.07	11 ± 0.46	11.1 ± 0.34
CYP-0.06 mg/kg bw	0.31 ± 0.06a	0.53 ± 0.06	10.9 ± 0.28	10.5 ± 0.43
CYP-0.12 mg/ bw	0.2 ± 0.03b,d	0.5 ± 0.06	11.5 ± 0.27a	10.9 ± 0.36
CYP-0.30 mg/kg bw	0.3 ± 0.04a	0.4 ± 0.06a	11.2 ± 0.3	11.0 ± 0.31
CYP-0.60 mg/kg bw	0.34 ± 0.04	0.51 ± 0.07a	11.8 ± 0.51a	10.5 ± 0.27
CAR-0.48 mg/kg bw	0.41 ± 0.06	0.51 ± 0.07a	8.9 ± 0.35a,d	9.6 ± 0.39a,d
CAR-0.96 mg/kg bw	0.5 ± 0.06a	0.56 ± 0.06a	10.4 ± 0.38	9.9 ± 0.33d
CAR-2.4 mg/kg bw	0.4 ± 0.07	0.6 ± 0.08b	11 ± 0.34	13.8 ± 0.35a,d
CAR-4.8 mg/kg bw	0.51 ± 0.06a	0.7 ± 0.08b,d	9.9 ± 0.36c	8.71 ± 0.42a,d
CYP-0.06 + CAR-0.48 mg/kg bw	0.4 ± 0.06	0.4 ± 0.03a	9.8 ± 0.24d	11 ± 0.27
CYP-0.12 + CAR-0.96 mg/kg bw	0.5 ± 0.06a	0.44 ± 0.04a	10 ± 0.32a,c	10.2 ± 0.21c
CYP-0.30 + CAR-2.4mg/kg bw	0.54 ± 0.04a,c	0.5 ± 0.06	10.5 ± 0.32	10.6 ± 0.29
CYP-0.60 + CAR-4.8mg/kg bw	0.6 ± 0.03b,d	0.6 ± 0.06a	9.8 ± 0.33a,d	10.2 ± 0.57c

Values are mean ± SEM from 6 rats in each group.

^aP < 0.05,

^bP < 0.01 and

^eP < 0.001: Compared between the control and treated groups.

^cP < 0.05 and

^dP < 0.01: Compared between the treated groups. Data analysed by one-way anova followed by Dunnett test.

Table 5.

Rat serum phosphorous content and amylase enzyme activity in control and treated samples. Cypermethrin, carbendazim and cypermethrin + carbendazim (each) at 12- and 24-h duration

	Phosphorous (mg/dl)		Amylase (IU/mg protein/ml × 10−2)
Groups	12 h	24 h	12 h	24 h
CONTROL	6 ± 0.26	6 ± 0.12	1.89 ± 0.1	1.19 ± 0.19
CYP-0.06 mg/kg bw	5.3 ± 0.21a	5.4 ± 0.22	1.92 ± 0.23	1.26 ± 0.15
CYP-0.12 mg/kg bw	6.01 ± 0.32	5.5 ± 0.17	1.87 ± 0.17	1.26 ± 0.15
CYP-0.30 mg/kg bw	6.8 ± 0.44a	6.1 ± 0.24	1.65 ± 0.13a	1.25 ± 0.16
CYP-0.60 mg/kg bw	7.5 ± 0.29a,b	6 ± 0.3	1.73 ± 0.15	1.25 ± 0.12
CAR-0.48 mg/kg bw	6.5 ± 0.32	7 ± 0.26a,b	1.63 ± 0.14	1.16 ± 0.11
CAR-0.96 mg/kg bw	5.93 ± 0.36	6.3 ± 0.19	1.77 ± 0.11	1.35 ± 0.11a
CAR-2.4 mg/kg bw	6.8 ± 0.25a	6 ± 0.27	1.83 ± 0.11	1.36 ± 0.12a
CAR-4.8 mg/kg bw	6.2 ± 0.37	7.6 ± 0.32a,b	1.76 ± 0.14	0.951 ± .09b
CYP-0.06 + CAR-0.48 mg/kg bw	6 ± 0.32	6.1 ± 0.32	1.91 ± 0.11	1.21 ± 0.1
CYP-0.12 + CAR-0.96 mg/kg bw	5.9 ± 0.28	5.9 ± 0.25	1.6 ± 0.14a	1.26 ± 0.15a
CYP-0.30 + CAR-2.4 mg/kg bw	6.9 ± 0.35b	5.1 ± 0.37b	1.62 ± 0.17a	1.14 ± 0.09
CYP-0.60 + CAR-4.8 mg/kg bw	5 ± 0.22a,b	6.5 ± 0.17	1.46 ± 0.1a,b	1.33 ± 0.13a

Values are mean ± SEM from 6 rats in each group.

^aP < 0.05,

^bP < 0.01 and

^eP < 0.001: Compared between the control and treated groups.

^cP < 0.05 and

^dP < 0.01: Compared between the treated groups. Data analysed by one-way anova followed by Dunnett test.

Alanine transaminase- and AST-specific activities were recorded for the corresponding time points following pesticide treatment (Table 6). ALT enzyme activity was significantly changed in a at time and dose-dependent manner. AST-specific activity fell into a low range of 0 to 0.5 IU/mg. Since control serum levels at the different time points varied, the effect of the individual compounds or their combination did not have meaningful impact (Table 6). Changes noted with regard to serum ALP-specific activity in relation to respective control were also seen. (Table 7). ALP enzyme activity was significantly changed in dose- and time-dependent manner. This ALP enzyme activity was changed significantly at 12 and 24 h, whereas GGT changed significantly only at 24 h. The level of rat serum GGT-specific activity was extremely low, and therefore, the variation noted with reference to control was not considered physiologically significant (Table 7).

Table 6.

Rat serum alanine transaminase (ALT)- and aspartate transaminase (AST)-specific activities in control and treated samples. Cypermethrin, carbendazim and cypermethrin + carbendazim (each) at 12- and 24-h duration

	ALT (IU/mg protein/ml × 10−2)		AST (IU/mg protein/ml × 10−2)
Groups	12 h	24 h	12 h	24 h
CONTROL	0.351 ± .028	0.163 ± 0.01	0.520 ± 0.07	0.349 ± 0.02
CYP-0.06 mg/kg bw	0.351 ± 0.01	0.193 ± 0.02a	0.564 ± 0.04	0.442 ± 0.02a,c
CYP-0.12 mg/kg bw	0.365 ± 0.02	0.159 ± 0.01	0.542 ± 0.03	0.437 ± 0.02a,d
CYP-0.30 mg/kg bw	0.371 ± 0.02a	0.181 ± 0.01b	0.544 ± 0.04	0.446 ± 0.04a,d
CYP-0.60 mg/kg bw	0.353 ± 0.02	0.183 ± 0.01b	0.531 ± 0.03	0.422 ± 0.03a,d
CAR-0.48 mg/kg bw	0.281 ± 0.02b,d	0.169 ± 0.01	0.238 ± 0.03b,d	0.325 ± 0.02a
CAR-0.96 mg/kg bw	0.301 ± 0.02a	0.159 ± 0.01	0.485 ± 0.03a	0.232 ± 0.03b,d
CAR-2.4 mg/kg bw t	0.343 ± 0.02a,c	0.162 ± 0.01a	0.594 ± 0.04b	0.226 ± 0.02b,d
CAR-4.8 mg/kg bw	0.307 ± 0.01b,c	0.179 ± 0.01b	0.471 ± 0.04b	0.260 ± 0.02b,d
CYP-0.06 + CAR-0.48 mg/kg bw	0.239 ± 0.02b,d	0.168 ± 0.01a	0.549 ± 0.04	0.376 ± 0.03a
CYP-0.12 + CAR-0.96 mg/kg bw	0.248 ± 0.02b,d	0.165 ± 0.01	0.232 ± 0.02b,d	0.359 ± 0.03a
CYP-0.30 + CAR-2.4 mg/kg bw	0.284 ± 0.01b,d	0.187 ± 0.02b	0.260 ± 0.02b,d	0.383 ± 0.02a
CYP-0.60 + CAR-4.8 mg/kg bw	0.307 ± 0.01b,c	0.168 ± 0.02	0.137 ± 0.01e,d	0.345 ± 0.03

Values are mean ± SEM from six rats in each group.

^aP < 0.05,

^bP < 0.01 and

^eP < 0.001: Compared between the control and treated groups.

^cP < 0.05 and

^dP < 0.01: Compared between the treated groups. Data analysed by one-way anova followed by Dunnett test.

Table 7.

Rat serum alkaline phosphatase ALP- and γ-glutamyl transpeptidase (GGT)-specific activities in control and treated samples. Cypermethrin, carbendazim and cypermethrin + carbendazim (each) at 12- and 24-h duration

	ALP (IU/mg protein/ml × 10−2)		GGT (IU/mg protein/ml × 10−2)
Groups	12 h	24 h	12 h	24 h
CONTROL	0.829 ± 0.04	0.605 ± 0.05	0.0159 ± 0.0007	0.013 ± 0.0006
CYP-0.06 mg/kg bw	0.903 ± 0.04a	0.714 ± 0.04b	0.0213 ± 0.0006a,d	0.0184 ± 0.0005a,d
CYP-0.12 mg/kg bw	0.511 ± 0.04e,d	0.803 ± 0.04b,d	0.0162 ± 0.0006	0.0154 ± 0.0004d
CYP-0.30 mg/kg bw	0.858 ± 0.04	0.768 ± 0.04b,d	0.0146 ± 0.0006	0.014 ± 0.0007a
CYP-0.60 mg/kg bw	0.828 ± 0.07	0.744 ± 0.05b,a	0.0182 ± 0.0007d	0.017 ± 0.0009a,d
CAR-0.48 mg/kg bw	0.561 ± 0.03d	0.597 ± 0.03a	0.0167 ± 0.0005	0.010 ± 0.0005a,d
CAR-0.96 mg/kg bw	0.600 ± 0.03b,d	0.660 ± 0.04b	0.024 ± 0.0005a,d	0.011 ± 0.0007a,d
CAR-2.4 mg/kg bw	0.609 ± 0.04b,d	0.680 ± 0.05a	0.0173 ± 0.0006	0.014 ± 0.0007a
CAR-4.8 mg/kg bw	0.792 ± 0.04b	0.770 ± 0.05b,d	0.0191 ± 0.0008d	0.0089 ± 0.0006b,d
CYP-0.06 + CAR-0.48 mg/kg bw	0.793 ± 0.04a	0.686 ± 0.04b	0.0157 ± 0.0004	0.0202 ± 0.0010a,d
CYP-0.12 + CAR-0.96 mg/kg bw	0.692 ± 0.03b,d	0.703 ± 0.06b	0.0165 ± 0.0006	0.0222 ± 0.0007a,d
CYP-0.30 + CAR-2.4 mg/kg bw	0.689 ± 0.04b,d	0.695 ± 0.04a	0.0232 ± 0.0009a,d	0.0198 ± 0.0008a,d
CYP-0.60 + CAR-4.8 mg/kg bw	0.769 ± 0.04a	0.660 ± 0.07a	0.0152 ± 0.0004	0.0158 ± 0.0005d

Values are mean ± SEM from 6 rats in each group.

^aP < 0.05,

^bP < 0.01 and

^eP < 0.001: Compared between the control and treated groups.

^cP < 0.05 and

^dP < 0.01: Compared between the treated groups. Data analysed by one-way anova followed by Dunnett test.

Discussion

α-cypermethrin is known to undergo metabolism through the cytochrome P450 microsomal system resulting in oxidative stress. The acute LD₅₀ value for α-cypermethrin in DMSO is reported to be 145 mg/kg bw. NOEL for α-cypermethrin is reported to be 1.5 mg/kg bw/day in dogs. ADI for α-cypermethrin is ranged between 0 and 0.02 mg/kg bw (Codex Alimentarius Commission 2003). It was considered that the increase in protein content could be due to an increase in the rate of translation of protein. Studies involving serum proteins have noted decreased level of total protein in serum of young rabbits because of cypermethrin toxicity (Lakkawar et al. 2006). There is a claim that cypermethrin had no significant effect on total protein content (Aldana et al. 2001). There has been claim that only amylase and ALP enzyme activities were affected by α-cypermethrin, whereas other enzyme activities remained unaltered (Khurshid 2003). The fall in serum enzyme activity may be due to the inhibition of transcriptional rate, enhanced clearance rate and pH change. Reduction in serum may also be due to inhibition/induction of mono-oxygenase enzyme system (Dikic et al. 2012).

The serum probably reflected that there was relative overload of the compounds resulting in an ability to increase the rate of catalytic transamination process between cellular substrates. It is also possible that limiting co-enzymic availability within the cells contributed to the observed decrease in ALT activity at higher dose of pesticide. Cellular transamination can also be considered a detoxification process, as removal of amino groups rendered the substrates more soluble for utilization in the metabolic pool.

That cypermethrin induced an increase in specific activity of brain AST is indicates an augmented process of oxaloacetate formation from aspartate. Increase in ALT and AST and BUN levels in rats treated with 520, 560, 600 mg/kg benomyl for 7 days has been reported and is equivalent to the carbendazim initiated increase in these enzyme activities (Selmanoglu et al. 2001). The low serum AST-specific activity can be considered due to increased protein content in the serum, as enhanced protein content was noted during the study using cypermethrin, carbendazim and combination. AST increase in cypermethrin-treated animals may be due to more pronounced cell damage and leakage of inner cellular enzymes. In addition, cypermethrin is a lipophilic molecule that can easily pass through the cell lipid bilayer and damages its integrity (Manna et al. 2004).

Carbendazim increased AST, ALT and BUN levels in rats when treated with 520, 560 and 600 mg/kg of benomyl for 7 days (Selmanoglu et al. 2001). NOEL for carbendazim is 25 and 2.5 mg/kg bw in dog and rat, respectively, as well as 30 mg/kg bw in rat (teratology). ADI for carbendazim ranged between 0 and 0.01 mg/kg bw (FAO/WHO 1988). It is well known that the ALP activity was dephosphorylated in animal tissues. ALP is a hydrolase and a transphosphorylase in function associated with cell membranes (Onikienko 1963). Increase in the specific activity of this enzyme therefore suggested the existence of a greater dephosphorylation potential within the animal cell.

Groups of 10 male and 10 female Wistar rats that received diets containing carbendazim at a concentration of 0, 8, 40, 200, 1000 and 5000 mg/kg per day for 30 days showed a decrease in body weight initially, but weight gain was observed at the two higher doses. The surviving animals at this dose, however, showed leukopenia, siderosis in liver and kidneys and arrest of spermatogenesis. The NOAEL was 200 mg/kg bw/day on the basis of reduced body weight gain and inhibition of spermatogenesis at 10,000 ppm and above (Scholz & Weigand 1972). Male and female rats received diets containing carbendazim at a concentration of 0, 80, 400, 2000 or 10,000 ppm for 93 days. Half of the animals in each group were sacrificed, and the remaining rats were fed untreated diet for a 12–14 day recovery period. In turn, it was noted that their bw was reduced at the higher doses, but no effects were observed because of feed consumption. No clinical signs of toxicity were noted. One male rat exhibited small testes and atrophy of the seminiferous tubuli. The NOEL was 200 ppm equal to 163 mg/kg bw/day, on the basis of reduced body weight at 10,000 ppm (Scholz & Schultes 1973). In yet another study, groups of two male and two female juvenile beagle dogs were fed carbendazim at a dietary level of 0, 500 or 2500 ppm for 28 days. Liver weights increased in females fed 500 and 2500 ppm carbendazim, and the activity of serum glutamic-pyruvic transaminase and alkaline phosphatase was increased in both male and females at 2500 ppm. The NOAEL was 500 ppm, equal to 19–21 mg/kg bw/day, on the basis of liver toxicity at 2500 ppm (Til et al. 1971).

Eventhough, initially the cypermethrin was found to be ineffective in the various tissues with regard to ALP specific activity, the phosphorous content was increased at 12 and 24 h. Decrease in ALP activity was taken as an index for parenchymal damage (Onikienko 1963). GGT catalysed the transfer of a glutamyl moiety between peptide donors and amino acid/peptide acceptors (Meister et al. 1973). GGT was also involved in the transfer of amino acid across the cell membrane. Further, GGT had a role in glutathione metabolism, transferring the glutamyl moiety to various acceptor molecule including water, L-amino acids and peptides. Such a process results in the retention of the cysteinyl glycine that was considered to preserve intracellular homoeostasis during oxidative stress. Shukla et al. (1989) reported an increase in serum GGT levels: GGT values in rodents are usually very low (Kaneko et al. 1997).

Serum GGT exhibited low specific activity. Serum GGT levels of male and female rats that were treated with benomyl reported no significant change (Seda & Tulin 2005). Several inferences could be derived based on these observations. Where GGT-specific activity was found increased, all GGT-related molecular processes would be enhanced in the rat, as well as their resulting metabolic consequences. It is possible that even though the rate of transpeptidation would become enhanced, the more significant aspect of increased GGT activity would be borne by leukotriene metabolism. GGT enzyme activity was increased in a dose and time dependant manner following alpha-naphthylisothiocyanate administration and a short-term relationship between increase in ductular cellularity and increased serum GGT activity has been reported (Richards et al. 1982). However, these results support our findings, since the time-dependent observations using the pesticides were limited to 24 h.

Amylase acts on complex carbohydrate-like starches to yield individual glucose units for energy metabolism within plant cells. Studies using cypermethrin had noted decrease in glycogen content at a given dose of 100 ppm, while at other doses such as 50, 200 and 400 ppm, the glycogen content remained increase in young chick embryos (Khurshid 2003). Changes in serum cholinesterase activity in the rat have been noted following the administration of cypermethrin in combination with chlorpyriphos within 24 h (Jadwiga et al. 2003). the control values occurred within two weeks.

The serum cholesterol content was two-fold higher compared to tissue cholesterol levels. For serum cholesterol, carbendazim was a more potent cause of increase than cypermethrin. The increase in serum cholesterol seemed reversible, since the combination of cypermethrin and carbendazim returned the serum cholesterol content to its normal level. Increase in urea content is indicative of an increase in ammonia generation. It was cited earlier that these pesticides enhanced the tissue transamination processes mediated by ALT and AST enzyme activities. It is therefore possible that increase in urea content is the consequence of metabolic changes brought about by these pesticides. Increase in uric acid content in the serum is an indication of an increase in purine metabolism that remains targeted by these pesticides. Khurshid (2003) reported increase in uric acid content attributable to sublethal doses of permethrin and cypermethrin in the chick, although increase in uric acid was noted within a time duration of 24 h.

With regard to serum glucose content, increase in glucose level was noted that possibly reflected increased glucose mobilization through breakdown of dietary or reserve complex carbohydrates. Increase in glucose level can also be caused by carbendazim (Selmanoglu et al. 2001). Changes in serum albumin content indicated either increased albumin synthesis by the tissues or decreased albumin degradation within each tissue. The probability of albumin synthesis by the various rat tissues contributing to an increase in serum albumin content can be considered as more realistic. Nevertheless, decreased content of protein in serum had also been reported (Lakkawar et al. 2004). A preferential depression of total serum proteins in female rats because of carbendazim administration had also been cited earlier (Selmanoglu et al. 2001). Increase in serum creatinine level occurred with the higher concentration of the pesticide combination, as well as carbendazim alone. It suggested an increase in creatine levels indirectly. Increase in creatinine level had been reported in rats, given 600 mg/kg per day of carbendazim (Selmanoglu et al. 2001). Although increase in AST activity level was higher in the pesticide-treated rats and AST is a known index for the development of polymyositis, in the absence of any report on increased creatine kinase activity in the serum, it cannot be stated firmly that increase in the creatine content because of the pesticide effect would lead to polymyositis in the experimental rats. Increase in serum calcium content occurred only with carbendazim, which also occurred, 24 h following the administration of the compound. However, phosphorous level registered an increase in cypermethrin and carbendazim from 6 to 24 h. This suggests that phosphorous mobilization might have occurred as a result of a shift in the Ca:P ratio in bone which might lead eventually to demineralization of bone matrix.

Conclusion

This study demonstrated the effect of low-dose cypermethrin, carbendazim and their combination on biochemical markers in male albino rat serum. The biochemical parameters such as serum total protein, glucose, cholesterol, urea, uric acid, albumin, calcium, phosphorous and creatinine were changed, following treatment. On the other hand, serum marker enzymes such ALT, AST, ALP, GGT and amylase were also altered following the administration of individual and combined form of pesticides. The experimental results suggested that even low-dose administration of these pesticides both individual and combined form has negative consequential effects on cell function and metabolism.

Declaration of interest

The authors declare no conflict of interest.