Analysis and Toxicity of Plain (PMP) and Blended (PMT) Indian Pan Masala (PM)

Abstract

Objective:

Betel leaf combined with areca nut is known as betel quid pan masala (PM), and tobacco with areca nut, catechu and lime is pan masala (PMT) blended with gulkhand. These narcotics are popular among young and old individuals. A prima facia chemical analysis and a toxicity assessment of PM in mice were conducted to study the relationship between longtime consumption of PM and health hazards.

Materials and Methods:

Chemical analysis of different types of PM was done employing HPLC, GLC, AAS, ES, TLC, GCMS and sequential extraction for PAH, pesticides, metals and minerals, electrolytes, drugs and xenobiotics. Ethanolic PM extracts were tested by IP and PO routes in inbred Swiss mice.

Results:

PAH, which are known xenobiotics for pre-cancerous lesions, were significantly high (p<0.01) in Rajaniganda and Pan Parag Zarda. Isomers of DDT and BHC, which principally act on nerves and muscles, were also high (p<0.01) in PM. The enhanced metal and mineral content of PM results in massive oral fibrosis. There is a high level of narcotics in PM, especially nicotine, a potentially cancerous agent in the gastrointestinal tract.

Conclusion:

Experimental studies with different extracts of plain and blended PM in mice fed for 16 and 90 days revealed no effect on blood and organ weights (kidney, heart, spleen and liver), but we did observe attenuated testis. However, in the bone marrow of the mice, chromosomes were most affected in the mice fed PM-Zarda blend for 3 months. The chromosomal abnormalities included ploidy, loss, breaks, gaps, deletions and exchanges in ring chromosomes. The PM caused sperm head anomalies (narrow, blunt, triangular and banana shapes), and the sperm were irregular, amorphous, tailless and rudimentary, with the maximum effect among the groups fed PM for 3 months. Significantly higher levels (p<0.01) of testis glycogen, cholesterol and protein were found. The group fed for 16 days showed no change in red blood corpuscles (RBC), white blood corpuscles (WBC), hemoglobin and erythrocyte sedimentation counts.

Introduction

The chewing of betel quid without tobacco is a widespread habit in the Orient. It began long ago when Portuguese people blended betel with tobacco in 1600 and called it betel quid, which was later made with areca nut, betel leaf, catechu, lime and tobacco. High consumption of betel quid is linked with oral cancer [1–6]. For two decades, a powdered chewing mixture, sold commercially as pan masala (PM), has been in abundant use in India among non-smokers as well as smokers who are attempting to refrain from smoking and tobacco use. Areca nut, a major portion of PM, is clastogenic, genotoxic and carcinogenic in animals [2, 7–8]. Catechu, an ingredient of PM, causes hepatic irritation, hyperplasia of oral mucosa [9], dominant lethal mutations and chromosomal damage in the bone marrow cells of mice [10]. Cancer incidence in southeast Asian countries is due not only to smoking but also to chewing betel quid and PM [5]. Betel quid is a mixture with different proportions of catechu, lime, areca nut, tobacco with spices, coconut, sugar and gulkhand (rose leaves and sugar). The different types of betel quid with certain addictive commercial products are sold in convenient one-dose sachets with familiar, popular brand names. PM is widely consumed by young and old individuals as a stimulant to remain anorectic and to compensate for a heavy extra workload; it is a mood elevator that provides relief from misery and grief. India and its neighboring countries are now in focus. Assam has the highest rates of cancers of the lip, buccal mucosa and pharyngeal areas due to the use of pan, betel nut and tobacco in betel quid or PM [11–12]. The tribal practice of reverse smoking with smokeless tobacco causes precancerous lesions [13], predominantly at the oral sites involved in squeezing and sucking the tobacco [14]. The proportions of the ingredients in PM differ by region. Assamese people incubate betel nuts with fibrous pericarp pits for 4 months and then strip the pericarp and use the endosperm (tamul). Gujarathi people make mava with dried betel nuts and lime, with or without tobacco. Raw kennel betel nuts, lime and tobacco are known as KWMP in Meghalaya. South Indians use betel leaves, nuts, lime and tobacco. Currently, northern Indians and people in the rest of the country consume PM. Worldwide, over 200 million people chew betel quid [15]. Juveniles may use as many as 30–40 betel quid per day, which may lead to the development of buccal cancer, stomatitis and precancerous lesions. Many areas, such as the tongue, alveolus, buccal mucosa, hard palate, tonsils, oropharynx, larynx and esophagus are affected by chewing PM and smoking tobacco, which may act synergistically to cause leukoplakia [16], submucous fibrosis and cancers of the oral cavity, oropharynx, larynx and esophagus. The habit of chewing PM is relatively new and occurs all over the world, but it is most prevalent on the Indian sub-continent. The use of PM is gaining social acceptance, and consumption is also increasing, with an increase in the sales of PM products every day. The habit of PM chewing is also slowly gaining a foothold in Western countries.

A preliminary study of Indian PM showed 13 polycyclic aromatic hydrocarbons (PAHs), DDT (2,2,bis-p-chlorophenyll,l,l-trichloroethane) and BHC (benzene hexachloride) isomers. In addition, there were nitrosamine, toxic metals (lead, cadmium, nickel) and pesticide residues in PM reported to be unsafe. The genotoxic potential of PM constituents is reflected by an increase in the rate of sister chromatid exchange (SCE) and the chromosomal aberrations in mouse bone marrow cells from the Chinese hamster ovary (CHO) cell line [17]. Aqueous [18] and ethanol [19] extracts of different PM brands showed mutagenic potential. Thus, a preliminary report indicates that PM has carcinogenic, tumorigenic, teratogenic and mutagenic potential [20]. Therefore, we have analyzed different brands of PM and the toxicology of PM effects in pure inbred Swiss mice.

Materials and Methods

The study design includes two parts: A) the chemical portion and B) the experimental portion.

Chemical Studies

Polycyclic aromatic hydrocarbons (PAHs)

Different brands of PM were dissolved in various solvents. Standard samples of different PAHs and relevant standards were procured for the high-pressure liquid chromatography (HPLC) analysis. A comparison of the PAH levels (in ug/g) in PM to standard values was made by performing a concomitant test using HPLC.

Isomers of DDT and BHC

Various PM samples were extracted and analyzed using conventional methods of gas-liquid chromatography (GLC) in comparison with standards. The results are expressed in parts per million (PPM) or ug/gm of PM.

Atomic Absorption Spectrophotometer Analysis (AAS)

Different brands of PM (0.5 gm) were placed in flasks; 5 mL of double distilled water (DDW) and 15 mL of concentrated nitric acid (HNO₃) + 0.5 mL of concentrated sulfuric acid (H₂SO₄) were added and held overnight at room temperature. Next, the samples were dried in a sand bath until they became a black cherry color. They were processed by adding perchloric acid and nitric acid at a 2:1 ratio until the samples turned yellow, and a 10 mL sample was used for analysis.

Emission Spectroscopy (ES) for metal analysis

An ultraviolet (UV) spectrograph Q24 instrument with a slit attachment including condenser Q was used. The slit was correctly set on the plate holder. The exposure wavelength scale was adjusted. The spectrograph image was adjusted from time to time, and the illumination of the camera lens was conducted according to the manufacturer’s instructions. With the proper condenser distance, UV spectra were obtained to measure the elements by using a Fun Ken spectrum control plate.

Analysis of Narcotic Substances

Conventional TLC (thin-layer chromatography) was used. The spots obtained using TLC were compared with a standard sample for evaluation.

Gas Chromatography and Mass Spectroscopy (GCMS)

Five organic solvents were used: petroleum ether (60.80 grades), ethyl acetate and petroleum ether in the ratio of 20:1 on toluene, chloroform, acetone and ethanol 95%.

Extraction of PM

Sequential extraction was carried out with various organic solvents according to their polarity so that the compounds present in the material were extracted completely. The following steps are involved in sequential extraction:

1. The material was dried completely at 40–50^°C in a hot air oven before extraction.

2. The material was ground to a 40 mesh-powder.

3. The material was weighed (10 g of powder) and transferred into a conical flask (500–1000mL).

4. The weighed material was macerated for 20–24 hr in various organic solvents at a 1:10 ratio in a constant sequence that was based on the polarity of the solvents. The following sequence of solvents was used: a) Petroleum ether (60–80^°C) b) Benzene/Toluene, c) Chloroform, iv) Acetone and v) Ethanol (95%).

5. After 20–24 hr, the macerated material was filtered with Whitman filter paper #1. The extract was collected by complete vacuum evaporation in a Rota vaporizer.

6. The residue was collected from the filter paper again and macerated in the same solvent for 20–25 hours. Processing was repeated three times with each solvent. The three extracts of one solvent were pooled in the same evaporating dish.

7. The solvent was changed after the material was fully dried.

8. The dried extract of each solvent was stored in an airtight bottle for analysis by GCMAS.

Determination of the volatile oil content of PM

1. Volatile oil extraction was performed by distillation using Clevenger’s apparatus.

2. Fifty grams of PM in 600 mL distilled water with a few pieces of porcelain in a distillation flask were connected to a Clevenger’s apparatus and kept on a heating mantle.

3. The graduated receiver was filled with water with no air bubbles.

4. Distillation was performed for 4–5 hrs at a rate that kept the lower end of the condenser cool.

5. The distillate was collected in the graduated receiver. Volatile oil, because it is light, remains in the upper layer.

6. The volatile oil was measured in the graduated receiver tube and % v/w was calculated on a dry weight basis.

The oil was collected in a small vial, and sodium sulfate was added to adsorb any water and moisture so that the pure oil remained for storage in a refrigerator (From 50 g PM, 0.6 ml volatile oil was obtained (1.2% v/w)).

Results

Levels of Polycyclic aromatic hydrocarbons

PAH was estimated with Manik Chand Sada and with Zarda, and the values were compared to common brands of PM (Table 1).The highest quantities of PAH were observed in Rajnigandha, Pan Parag and Zarda, suggesting that they can produce ill effects in individuals who regularly consume PM. Furthermore, some of the PAHs are known to be potent carcinogens, and regular consumption of them can even lead to the development of cancer in the mouth, esophagus, stomach or other parts of the body.

Table 1.

Levels of Polycylic Aromatic Hydrocarbons (PAH) in pan masala (ppm or µg/gm)

Compound	a	b	c	d	e	f	g	h	i
1 Napththalene	0.75	0.59	0.39	0.33	0.99	13.99	0.66	1.04	1.07
2 Acenaphthylene	0.06	0.41	0.07	0.38	0.06	12.06	0.19	0.07	0.06
3 Acenaphthene	0.05	ND	0.05	0.13	0.05	0.05	ND	0.05	0.05
4 Fluorene	0.06	ND	0.20	0.48	0.06	ND	ND	0.13	0.13
5 Phenanthrene	0.06	0.13	0.07	0.08	0.06	0.15	0.03	0.05	0.04
6 Anthracene	0.01	0.01	0.01	0.11	0.01	0.11	0.04	0.01	0.01
7 Fluoranthene	0.09	0.07	0.07	2.21	0.07	1.61	0.17	0.04	0.04
8 Pyrene	1.02	0.38	0.60	11.32	2.01	13.50	2.89	0.61	0.58
9 Chrysene	0.08	0.13	0.07	0.06	0.08	ND	0.13	0.08	0.08
10 Benz (Q) pyrene+perylene	0.03	0.02	0.03	0.04	0.03	0.04	0.08	0.04	0.04
11 Benz(b) Fluoranthene	0.04	0.04	0.05	0.03	0.04	0.07	0.08	0.06	0.06
12 Benz(k) Fluoranthene	0.09	0.06	0.11	0.09	0.07	0.07	0.10	0.11	0.12
13 Benzo(a)pyrene	0.03	0.04	0.02	0.07	0.04	0.10	0.07	0.04	0.04
14 PAH Total	2.37	1.81	1.77	15.39	3.51	41.75	4.71	2.33	2.29

a: Pan Parag Sada, b: Santoor Sada, c: Nawabi Masala, d: Rajnigandha Sada, e: Manikchand Sada, f: Pan Parag Zarda, g: Santoor Zarda, h: Pan King Zarda, i: Manik Chand Zarda

Levels of HCH and DDT

The most common brands of PM were evaluated for the presence of HCH, DDT and their isomers. The data suggest the presence of different isomers of DDT and HCH, and the total quantity was higher than permissible limits (Tables 2a and 2b). Some of the samples, such as Santoor, contained a very high concentration of HCH. Both these pesticides are persistent in nature and remain for a long period in human bodies, and they can have adverse effects on health, including neurotoxic symptoms in exposed persons. The new samples we analyzed contained levels of pesticides similar to those seen in our earlier studies.

Table 2.

(a) Pesticide concentration (pm or μg/gm) in pan masal (sada)

Compound	a	b	c	d	e	f	g
HCH
Alpha HCH	0.061	2.546	0.047	0.090	0.52	0.56	0.070
Gamma HCH	0.075	11.544	1.075	0.045	0.045	0.075	0.060
Beta HCH	0.055	13.997	0.319	0.077	0.077	0.092	0.080
Total HCH	0.191	28.087	1.441	0.211	0.211	0.223	0.210
DDT
P,p′DDE	0.009	0.010	0.008	0.006	0.007	0.016	0.012
O,p′DDT	0.010	0.027	0.012	0.007	0.010	0.024	0.010
P,p′DDT	0.027	0.063	0.030	0.015	0.026	0.060	0.028
Total DDT	0.046	0.100	0.050	0.028	0.043	0.100	0.050

a: Pan Parag, b :Santoor, c: Nawabi Masala, d: Rajnigandha, e: Manikchan, f: Mahak, g: Chutk

Table 2.

(b) Pesticide concentration (ppm or μg/gm) in pan masala (zarda)

Compound	a	b	c	d	e	f
HCH
Alpha HCH	0.024	0.069	0.058	0.026	0.032	0.040
Gamma HCH	7.650	31.450	0.959	6.640	7.420	7.120
Beta HCH	0.037	19.632	1.165	0.037	0.046	0.036
Total HCH	7.711	51.151	2.182	6.703	7.798	7.196
DDT
p.p′ DDE	0.009	0.007	0.010	0.006	0.004	0.007
o.p′ DDT	0.010	0.007	0.027	0.009	0.009	0.008
p.p′ DDT	0.027	0.017	0.063	0.026	0.016	0.062
Total DDT	0.046	0.031	0.100	0.031	0.029	0.077

a: Pan Parag Zarda, b: Santoor Zarda, c: Panking Zarda, d: Manikchand Zarda, e: Mahak zarda, f: Chutki Zarda

Proximate analysis of PM samples with Atomic absorption spectrophotometer

Five Sada samples and 5 Zarda samples of common brands of PM were analyzed. All of the samples were free of cadmium and lead. In our earlier studies, cadmium, lead and nickel were found in traces, but the most interesting finding of the analysis was the presence of copper, zinc and magnesium in excessive amounts in all the samples. Magnesium, in particular, was found in excessive amounts (Table 3). Copper and magnesium are suspected to be the metals that most strongly stimulate the production of fibroblasts, leading to fibrotic changes in mucosal membranes. Cases of submucosal fibrosis in human beings are likely due to excessive levels of these minerals. The body has the capacity to absorb or excrete excess amounts of these metals, but it is likely that repeated exposure to these minerals could have other adverse effects on the body.

Table 3.

Proximate (direct) analysis of pan masala samples

	Manik Chand		Pan Parag		Chutki	Rajni-gandh	Mahak		Tulsi	Vimol
	Tobacco	With-out	Tobacco	With-out N.T.a	N.T.a	N.T.a	Tobacco	N.T.a	Tobacco	Tobacco
Cadmium (μg/gm)	N.D.b	N.D.b	N.D.b	N.D.	N.D.b	N.D.b	N.D.b	N.D.b	N.D.b	N.D.b
Lead (μg/gm)	N.D.b	N.D.b	N.D.b	N.D.b	ND.b	N.D.b	N.D.b	N.D.b	N.D.b	N.D.b
Cu (μg/gm)	21.6	17.60	21.44	19.68	5.92	19.36	14.56	13.28	9.60	15.68
Zn (μg/gm)	11.78	15.30	11.78	10.11	9.85	11.21	11.69	7.82	7.56	10.59
Mg (μg/gm)	7561.6	4345.89	3878.42	5321.42	19859.6	3642.12	11691.73	2688.64	7530.82	19501.0

^aNot Traceable,

^bNot detected

Emission Spectroscopic analysis of PM samples

Nearly 40 samples of various brands of PM commonly used in Ahmedabad, Gujarat were analyzed using emission spectroscopy (Tables 4a and 4b). The results suggest that various metals are present (magnesium, copper, sodium, calcium, aluminum, potassium, manganese, silica, etc.) Continuous use of these brands of PM and the presence of these metals in excess may disturb the balance of these metals in the body, with diverse effects on both the individuals who use these brands regularly and those who use them in excess daily.

Table 4.

(a) Analysis of pan masala samples

Name of the sample	Mg	Cu	Na	Ca	Al	K
Vazin Gutkha (Tobacco)	++ (Excess)	+ (Present)	+	++	Traces	+
Kisan Gutkha (Tobacco)	++	+	+	++	-	+
Dawat Gutkha (Tobacco)	++	+	+	++	-	+
Zat Pat Gutkha (Tobacco)	++	+	+	++	-	+
Soni Gutkha (Tobacco)	++	+	+	++	-	-
Kuber Gutkha (Tobacco)	++	Traces	Traces	++	-	-
Mirad 2000 (Tobacco)	+	-	-	++	-	-
Budhlane (Tobacco) Raju Supari (Plain)	++	FT (Freely traceable)	+	++	-	FT
Raju Supari (Plain)	++	Traces	++	++	-	Traces
Dillagi Supari (Plain)	++	++	-	++	Traces	++
Plain Kacchi Supari
Plain Kacchi Supari	+	FT	-	++	Traces	-
Saheli Sweet Supari (Plain)	++	-	-	++	Traces	-
Poonam Supari (Plain)	++	Traces	-	++	Traces	-
Simple Roasted Supari (Plain)	++	-	-	++	-	-

+ Present but could not quantified

- Nil

Table 4.

(b) Analysis of pan masala samples

Name of the sample	Si	Mn	Mg	Cu	Na	Ca
Manikchand Gutkha	-	-	+	FT (Freely treaceable)	FT	+
Tulsi Mix	-	-	+	FT	FT	+
Manikchand Plane	Traces	Traces	+	+	Traces	+
Rajnigandha Plain	+ (Present)	Traces	+	++	Traces	+
Pan Parag Tobacco	-	Traces	+	FT	Traces	+
Pan Parag Plain	-	-	+	FT	FT	+
Mahak Tobacco	-	-	+	FT	FT	+
Mahak Plain	Traces	-	+	FT	-	+
Moolchand with Tobacco	-	-	+	Traces	Traces	+
Tara Gutkha Tobacco	-	-	+	Traces	FT	+
Vimal Gutkha (Tobacco)	Traces	-	+	FT	FT	+
Pan King (Tobacco)	-	-	+	-	FT	+
Kuber Khaini (Tobacco)	-	-	+	-	Traces	+
Katha Paste	-	-	+	-	FT
Janta Banarasi Brand	Traces	Traces	-	-	-	-

Qualitative estimation of narcotic substances and cannabis using the TLC method

The 10 most common varieties of PM were evaluated for charas, brown sugar, morphine and nicotine (Table 5). Nicotine was present in excess in all of the samples, but no traces of narcotics or cannabis were present in any sample. However, the adverse effects of excessive nicotine in the body are known. It produces nausea in exposed persons, and it may also lead to the development of certain types of cancer in the gastrointestinal tract.

Table 5.

Qualitative estimation of narcotic and cannabis substances in pan masala

Name of the sample	Charas	Brown Sugar	Diacetyl Morphine	Morphine	Papavarine	Opium	Nicotine
Manikchand Gutkha (Tobacco)	-	-	-	-	-	-	+ (Present)
Pan Parag utkha (Tobacco)	-	-	-	-	-	-	+
Dawat Gutkha (Tobacco)	-	-	-	-	-	-	+
Mahak Gutkha (Tobacco)	-	-	-	-	-	-	+
Tulsi Mix Gutkha (Tobacco)	-	-	-	-	-	-	+
Moolchand Gutkha (Tobacco)	-	-	-	-	-	-	+
Som Gutkha (Tobacco)	-	-	-	-	-	-	+
Vazir Gutkha (Tobacco)	-	-	-	-	-	-	+
Vimal Gutkha (Tobacco)	-	-	-	-	-	-	+
Zat Pat Gutkha (Tobacco)	-	-	-	-	-	-	+

Experimental Studies

This experimen tal study was conducted with the approval of the local ethics committee. Pure inbred Swiss male mice were used for the experimental studies. The animals were exposed to PM mixed with food through ip and by oral intubation. In the short-term studies, primarily male Swiss mice were used. The aqueous and solvent (Dimethyl Sulfoxide; DMSO) extracts of the Manik Chand variety of PM with and without Zarda were prepared for exposure through the ip route.

Preparation of ethanol PM extract

Powdered Manik Chand brand of PM was mixed with 5 volumes of redistilled ethanol, and the contents were extracted by agitation using a rotary shaker overnight at room temperature. The supernatant collected after centrifugation at high speed in an ultracentrifuge was evaporated until dry. The dry residue was dissolved again in redistilled ethanol to obtain the desired dose of original PM powder.

Preparation of food with 2% PM

Manik Chand PM with or without Zarda was turned into powder using a mixer. A 100-g portion of this fine PM powder was mixed thoroughly with 4900 g of feed given to the mice routinely. Mice usually eat 5 g of feed per day; thus, 100 mg of PM was consumed by the experimental animals daily. Discrete biochemical changes in the testis were studied in mice by orally administering 3 different dose levels (i.e., 30, 40, 100 mg/kg b.w. of PMT extract) and a placebo (0.5 mL olive oil) for a period of 60 days using 4 sets of mice (n=10).

Animals and Treatments

Pure inbred Swiss male mice (and female mice in the chronic toxicity studies) 6 to 8 weeks old were used throughout the studies. Animals of the same sex were housed in a cage in an air-conditioned animal house (constant humidity 55.6%; 12-hr light/dark cycle) and were provided with food prepared in the Institute. The composition of the food was 70% wheat, 20% cracked Bengal gram, 5% fishmeal, 4% yeast powder and 1% shark liver oil in the form of dry mesh, and water was given ad libitum. A group of animals were exposed by the ip route through oral intubations and PM mixed into their feed. Through oral intubations, the animals were given the scheduled dose 5 days each week, whereas PM mixed in feed was given daily. The institutional ethical committee for animals approved the experimental protocols and the maintenance of the animals and their usage during this study.

Toxicological evaluation through ip and po

The aqueous and solvent (DMSO) extracts of the Manik Chand brand of PM with and without Zarda were prepared. A preliminary experiment to evaluate the toxic dose level was initiated by exposing mice to doses of 0.2, 0.5, 1.0 and 2 g/kg bwt PM through the ip route. The animals could not tolerate doses of 0.5 g/kg b wt and above; therefore, the experimental animals were actually exposed to 8, 40 & 200 mg/kg bwt. One group (a) was exposed for two weeks ip, and another group was exposed through oral feeding for three months. Distilled water (0.1 mL) or DMSO (02 mL) was administered as a negative control, while benzo[a]pyrene or trichloroacetic acid was used as a positive control. After scheduled ip exposure, only histopathological and hematological evaluation could be done, whereas in the group exposed for 3 months, histopathological hematological cytogenetic and spermatological studies were performed. (b) Another group of animals received ethanol PM extract through oral intubations for ninety days. Special emphasis was placed on examining testicular tissue, and biochemical parameters, such as glycogen, cholesterol and protein, were estimated in testicular tissue. Histopathological alterations and the weights of different organs were also recorded. (c) For the chronic toxicity studies, PM with and without Zarda was mixed with feed and fed to the animals routinely.

This experiment offered a direct and effective way to identify the chemicals responsible for spermatogenic damage in rodents and men. The exposed animals were euthanized after two weeks or three months by cervical dislocation, and the cauda epididymis was removed. Suspensions were prepared from each cauda by mincing the cauda in 4 mL phosphate buffer saline (pH 6.8) and allowing the mixture to stand for 15 minutes at room temperature. Tissue fragments were removed, and an aliquot (0.5 ml) of the sample was mixed (10.1) with 1% aqueous eosin. Smears were made 30 minutes later from the stained sperm suspension. The slides were allowed to dry in air and mounted with per- mount. Ten slides were prepared from each mouse (5 slides per cauda), and the head shape morphology of 100–200 sperm (1000–2000 sperm per mouse) was examined under the light microscope at 400-fold magnification. Different organ weights were also recorded. The data were analyzed for statistical significance by applying Student’s t test and the X² test.

Bone marrow metaphase assay

The animals were injected with colchicine solution 1.5 hr prior to bone marrow sampling (4 mg colchicine/kg b/ wt). Bone marrow was flushed in HBSS, centrifuged for five minutes. The supernatant was discarded, and then hypotonic solution (1% sodium citrate) was slowly added to dispense the pellet. The mixture was centrifuged again after hypotonic treatment cells were fixed to the re-suspended pellet in a freshly prepared cold methanol/acetic acid mixture (3:1). The slides were stained with Giemsa 5% for 10 minutes and then examined for any chromosomal abnormalities.

Biochemical estimation

The estimation of protein was done according to a previously reported method [21]. A known amount of liver tissue was homogenized in 5 mL of distilled water. To 1 ml of homogenate (which was obtained after centrifugation), 4 mL of biuret reagent was added. Distilled water was added in place of the homogenate. The tubes were kept at room temperature for 30 minutes. The resultant color intensity was recorded at 540 nm.

The protein concentration was determined using the following regression formula:

$$\text{X}=0.077+18.70\hspace{0.17em}(\text{y})\text{q}$$

Where

X

Concentration of protein in mg

Y

O D of unknown sample

$$\text{Calculation}:\text{Amount}\hspace{0.17em}\text{of}\hspace{0.17em}\text{protein}=\frac{\text{Concentration}\hspace{0.17em}\text{of}\hspace{0.17em}\text{Mean}}{\text{Tissue}\hspace{0.17em}\text{weight}}\frac{\text{OD}\hspace{0.17em}\text{Dilutrin}\hspace{0.17em}\text{X}\hspace{0.17em}100}{\text{Aliquot}\hspace{0.17em}\text{volume}}$$

Histopathological examination: After the animals were euthanized, different organs were fixed in 10% formal saline. Histopathology is currently being conducted using conventional methods, and the results will be reported at a later date.

Statistical analysis

Student’s t test was employed [22].

Chemicals

All the chemicals used were analytical grade products from Sigma-Aldrich Company with impurities ranging from 0.001% to 0.0001%.

Results

IP exposure to PM for 16 days

Hematological changes, including RBC and WBC counts, differential hemoglobin and ESR, were analyzed in groups exposed to different doses of PM and compared with positive and negative control data (Table 6). The data suggest that there is no significant change in the hematological values of the experimental groups compared to the controls.

Table 6.

Haematological findings intraperitoneal (IP) exposure to Manikchand pan masala in Swiss inbred mice

Groups	Units	R.B.C.f Million/mm3	W.B.C.g -/mm3	P	L	DC%h E	M	B	Hbi gm%	E.S.R.j mm
Negative control (NC)	0.1mL DMSOd	5.71±0.1	8682±140	N=6 47.5±8.81	N=6 51.83±8.94	N=6 0.83±0.17	-	-	13.42±0.54	9.25±1.19
Positive control (PC)	4 mg/kg b.wt BAP on 0.1mL DMSOe	5.09±0.2	8000±570.7	N=6 59.36±5.34	N=6 40.36±5.40	N=6 0.71±0.29	-	-	15.58±0.47	8.0±0.87
Pan Masala I (PMa)	40 mg/kg b wt	5.18±0.6	7300±475.6	N=6 32.7±1.69	N=6 67.0±1.63	N=6 0.2±0.13	-	-	16.1 ± 0.39	12.2±1.38
Pan Masala II (PMb)	200 mg/kg b wt	4.85±0.7	7400±507.2	N=4 63.0±2.08	N=4 37.2±2.08	N=4	-	-	13.33±0.37	5.58±0.30
Pan Masala III (PMc)	8 mg/kg b wt	5.14±0.2	8180±202.4	N=6 42.5±4.29	N=6 57.0±4.18	N=6 0.5±0.29	N=6	-	16.25±0.25	5.39+0.39

NC, PC, PM^a and PM^c = Each group 16 days exposure,

PM^b = 8 days exposure,

^deVehicle for NC and PC (n=6 mice/group) Each 6 weeks old. P=Polymorphs, L=Leucocytes, E=Eosinophils, M=Monocytes, B=Basophiles,

R.B.C.=Red Blood Corpuscles

W.B.C.=White Blood Corpuscles

DC=Differential Count

Hb=Hemoglobin

E.S.R.=Erythrocyte Sedimentation Rate

Oral Administration of PM for three months

After 3 months of PM administration, the animals’ final weights were measured and compared to their initial weights. Blood was collected directly from the heart, and then the animals were euthanized. Organ weights were recorded (Tables 7a and 7b). There were apparently no changes in organ weights compared to the controls. No significant changes were seen in the hematological parameters in the PM/sada group or the Zarda group when compared with the control group (Tables 8a and 8b).

Table 7.

(a) Body and organ weights of pan masala extract treated pure inbred Swiss mice

Treatment	Body weight (gm)		Testes (gm)	Liver (gm)	Heart (gm)	Kidney (gm)	Spleen (gm)
	Initial	Final
Group I (n=6) Intact control or vehicle treated	21.5	22.5	0.150	1.51	0.120	0.401	0.043
	22.0	23.4	0.164	2.07	0.140	0.430	0.097
	26.0	27.4	0.174	1.78	0.135	0.429	0.077
	20.5	22.4	0.168	1.62	0.129	0.400	0.052
	24.0	25.2	0.160	1.70	0.130	0.398	0.095
	25.0	26.1	0.196	1.58	0.120	0.500	0.072
Group II (n=6)	26.5	27.0	0.152	2.25	0.138	0.480	0.-066
Intact+Pan	23.5	24.6	0.146	1.78	0.129	0.420	0.043
Masala 20	27.0	28.2	0.164	1.90	0.150	0.410	0.102
Mg/kg b wt/day for 60 days	27.5	28.0	0.160	1.80	0.110	0.385	0.056
	24.0	25.8	0.164	1.65	0.145	0.400	0.074
	22.0	22.8	0.150	1.87	0.130	0.390	0.084
Group III (n=6)	28.0	29.0	0.146	2.31	0.132	0.407	0.191
Intact+Pan	24.5	25.4	0.164	2.18	0.119	0.415	0.096
Masala 40	24.0	25.0	0.150	1.90	0.105	0.400	0.069
Mg/kg b wt/day for 60 days	25.0	25.8	0.152	2.07	0.126	0.368	0.085
	22.0	23.2	0.160	1.87	0.136	0.368	0.138
	26.0	26.8	0.174	2.47	0.141	0.496	0.127
Group IV (n=6)	24.5	25.0	0.151	2.56	0.136	0.500	0.102
Intact+Pan	26.5	27.2	0.162	2.43	0.140	0.475	0.100
Masala 100	26.5	27.3	0.164	2.34	0.136	0.407	0.070
Mg/kg b wt/day for 60 days	24.0	26.0	0.141	2.40	0.149	0.419	0.090
	23.0	24.0	0.146	1.90	0.153	0.410	0.095
	22.0	23.4	0.140	1.78	0.120	0.429	-

Table 7.

(b) Body and organ weights of pure inbred Swiss mice after oral admini-stration of Manikchand pan masala for 3 months

Treatment	Body weight (gm)		Testes (gm)	Liver (gm)	Heart (gm)	Kidney (gm)	Spleen (gm)	Lungs (gm)
	Initial	Final
Control	24	28	0.168	1.51	0.120	0.401	0.043	0.180
	28	29	0.164	2.07	0.140	0.430	0.097	0.210
	26	28	0.174	1.78	0.135	0.429	0.077	0.205
	24	26	0.183	1.62	0.129	0.400	0.052	0.190
Manikchand Plain (8 mg)	26	28.2	0.132	1.70	0.130	0.398	0.956	0.200
	28	29.8	0.150	1.58	0.120	0.500	0.072	0.195
	26	28.2	0.169	2.25	0.138	0.480	0.066	0.188
	20	23.1	0.172	-	0.129	0.420	0.043	0.210
Manikchand Plain (40mg)	24	26.4	0.142	1.78	0.15	0.410	0.102	0.15
	26	27.8	0.140	1.90	0.11	0.385	0.056	0.19
	26	27.8	0.165	1.80	0.145	0.400	0.074	0.17
	28	27.6	0.140	1.65	0.130	0.390	0.084	0.19
Manikchand Plain (100 mg)	24	26.2	0.148	1.87	0.132	0.339	0.191	0.207
	30	31.4	0.176	2.31	0.119	0.357	0.096	0.183
	26	27.8	0.132	2.18	0.105	0.315	0.069	0.179
	24	25.8	0.150	-	0.126	0.407	0.085	0.190
Manikchand with Zarda (8 mg)	25	26.6	0.148	2.08	0.136	0.419	0.080	0.194
	26	28.0	0.143	2.02	0.140	0.405	0.100	0.199
	24	26.2	0.150	2.12	0.136	0.400	0.050	0.205
	25	27.1	0.162	1.98	0.117	0.038	0.900	-
Manikchand with Zarda (40 mg)	26	27.6	0.148	2.43	0.149	0.486	0.075	0.180
	28	29.4	0.134	2.01	0.162	0.496	0.090	0.175
	28	29.6	0.142	1.98	0.153	0.500	0.095	0.221
	24	25.8	0.162	1.96	0.120	0.475	0.800	0.221
Manikchand with Zarda (100 mg)	26	28.2	0.142	1.92	0.136	0.300	0.094	0.180
	28	29.2	0.136	1.86	0.140	0.419	0.056	0.135
	26	28.1	0.155	2.22	0.136	0.486	0.061	0.140
	28	29.2	0.149	2.42	1.49	0.475	0.075	0.160

6 Animals in each group. (n=6)

Table 8.

(a) Oral administration of pan masala to pure inbred Swiss male mice (Haematological findings-3 months)

Groups	Units	R.B.C. Million/mm3	W.B.C. -/mm3	P	L	DC% E	M	B	Hb Gm%	E.S.R. mm
Negative Control	0.4ml sterile water	5.71±0.1	8680±140	21.2±0.6	72.3±0.84	-	1.5±0.3	-	13.4±0.5	10.±0.7
Positive Control	25 mg/kg TCA b.wt	5±0.2	8000±460	19.7±3.2	81.3±0.99	0.33±0.2	1.2±0.3	-	15.5±0.5	11.3±1.9
Pan Masala With Sada I	8 mg/kg b.wt.	5.1±0.4	7300±475	26±1.17	71.3±1.87	0.17±0.1	2.5±0.4	-	13.6±0.4	7.7±1.6
Pan Masala With Sada II	40 mg/kg b.wt.	4.9±0.2	7100±407	27±1.13	70±1.24	0.33±0.2	2.7±0.3	-	13.3±0.3	9.4±0.48
Pan Masala With SadaIII	200mg/kg b.wt.	4.6±0.7	7050±202	26.4±3.6	70.67±3.6	1±3.6	1.3±0.4	-	12.2±0.2	8.5±0.43

(n=6 mice/group) Each 6 weeks old. P=Polymorphs, L=Leucocytes, E=Eosinophils, M=Monocytes, B=Basophiles, R.B.C.=Red Blood Corpuscles, W.B.C.=White Blood Corpuscles, DC=Differential Count, Hb=Hemoglobin, E.S.R.=Erythrocyte Sedimentation Rate

Table 8.

(b) Oral administration of pan masala to pure inbred Swiss mice (Haematological findings-3 months)

Groups	Units	R.B.C. Million/mm3	W.B.C. -/mm3	P	L	DC% E	M	B	Hb Gm%	E.S.R. mm
Negative control	0.4ml sterile water	5.71±0.1	6.97±0.6	28±2.6	71±2.5	-	0.7±0.3	-	13.4±0.5	10.±0.7
Positive control	25 mg/kg TCA b.wt	5±0.2	5.3±0.34	35.8±2.9	62.6±3.2	0.38±0.3	1±0.2	-	15.5±0.5	11.3±1.9
Pan Masala With Zarda I	8 mg/kg b.wt.	4.1±0.2	5±0.4	35±2.3	62.8±2.2	0.4±0.4	1.8±0.2	-	13.1±0.4	8.8±1.6
Pan Masala With Zarda II	40 mg/kg b.wt.	4.12±0.6	5.51±0.4	32.8±2.2	64.1±2.1	0.92±0.3	2.2±0.2	-	12.9±0.2	9.4±0.48
Pan Masala With ZardaIII	200mg/kg b.wt.	4.1±0.7	5±0.58	35±2.9	61.6±3.1	1.45±0.2	1.5±0.3	-	12.2±0.2	8.6±0.43

(n=6 mice/group) Each 6 weeks old. P=Polymorphs, L=Leucocytes, E=Eosinophils, M=Monocytes, B=Basophiles, R.B.C.=Red Blood Corpuscles, W.B.C.=White Blood Corpuscles, DC=Differential Count, Hb=Hemoglobin, E.S.R.=Erythrocyte Sedimentation Rate

Bone marrow chromosomes

After 2 weeks of ip exposure and 3 months of continuous oral exposure to PM with and without Zarda, there was a pronounced effect on the bone marrow chromosomes of the experimental mice when compared with the controls. The effect appeared to be more pronounced in the Zarda group; when compared with the plain PM group, there was a significantly higher prevalence of chromosomal abnormalities (Table 9). The changes observed were ploidy loss, breaks, gaps, deletions and exchanges, including ring chromosomes.

Table 9.

Effect of oral administration of Manikchand pan masala with and without zarda on the somatic (bone marrow) chromosomes of pure inbred Swiss mice after 2 weeks and 3 months exposure

Group	Dose (mg/kg b wt)	Chromosomal abnormalities (%)
		2 weeks	3 months
Control (Distilled water)	0.2 mL	1.08	2.20
Manikchand Plain	8	1.82	1.74
Manikchand Plain	40	3.62	3.88
Manikchand Plain	100
Manikchand with Zarda	8	4.20	5.64
Manikchand with Zarda	40	4.6	5.82
Manikchand with Zarda	100	5.8	6.60

6 Animals killed in each group (n=6)

bwt-body weight

p<0.01 (Contro verses test groups

Sperm head abnormalities: Compared to the controls, there were no significant differences in the organ weights of the kidneys, heart, spleen or liver. However, a slight reduction in testicular weight was observed. There was a slight (although non-significant) reduction in testicular tissue, but there were more pronounced changes in sperm head abnormalities, which included narrow, blunt, triangular, and banana shapes, irregular amorphous shapes, and tail-less and rudimentary sperm (Table 10). The effects were more pronounced in the group with 3 months of exposure.

Table 10.

Effect of oral administration of Manikchand Pan Masala on the sperm after 2 weeks and 3 months exposure

Group	Dose (mg/kg b wt)	Sperm abnormalities (%)
		2 weeks	3 months
Control (Distilled water)	0.2 mL	1.42	1.48
Manikchand Plain	8	1.56 NS	1.82 p<0.05
Manikchand Plain	40	1.82 p<0.05	1.96 p<0.05
Manikchand Plain	100	2.26 p<0.01	3.64 p<0.01
Manikchand with Zarda	8	2.18 p<0.01	2.62 p<0.01
Manikchand with Zarda	40	2.62 p<0.01	2.88 p<0.01
Manikchand with Zarda	100	3.82 p<0.001	4.66 p<0.001

6 Animals in each group

p<0.01 Significant (Significance: Control versus test group)

p<0.001 Highly Significant

NS: Not Significant

Biochemical findings

When biochemical parameters, including glycogen, cholesterol and protein, were evaluated in testicular tissue, there were highly significant changes in glycogen, cholesterol level and protein content. This effect appeared to be more pronounced in the group that consumed more PM (Table 11). After 60 days of PMT exposure, glycogen and cholesterol in the testis showed significant deterioration at all the dose levels compared with the controls, although the effect was not dose-dependent. Protein, although significantly higher in the 30-mg/kg group, was significantly lower at the 40- and 100-mg/kg dose levels compared with control (vehicle treated) mice. Discrete biochemical changes of the testis were studied in 4 sets of mice (n=10) that received orally administered PMT extract at 3 dose levels (i.e., 30, 40, 100 mg/kg b.w.) or a placebo (0.5 mL olive oil) for a period of 60 days.

Table 11.

Biochemical changes in testicular tissue after administration of pan masala exctract

Treatment (n=6)	Glycogen (mg/gm)	Cholesterol (mg/gm)	Protein (mg/gm)
Group I: Vehicle treated group	3.46±0.04	8.62±0.2	182.2±2.5
Group II: 30 mg/kg bwt/day for 60 days	1.82±0.04*	5.22±0.2*	1130.32±1.4*
Group III: 40 mg/kg bwt/day for 60 days	1.92±0.02**	5.12±0.12*	108.07±0.2**
Group IV: 100 mg/kg bwt/day for 60 days	1.80±0.01**	3.75±0.2**	120.64±2.2*

Mean±SEM of animals

N.S.=Non-significan Group II, III & IV were compared with I.

^*p<0.01-Significant

^**p<0.001-Highly significant

bwt-body weight

Discussion

The study determined the chemical components of plain and blended PM as well as its sub-acute and biologic effects on weight, blood, bone marrow and sperm. The chemical composition of PM was found to be responsible for its biochemical actions and toxicity. Currently, PM usage is spreading widely among youth, workers and executives because it is a more effective mood elevator than smoking and may also act as a central nervous stimulant, offering temporary relief from grief and exhaustion due to manual and non-manual stress. Many reports on PM are available, but there are no long-term experimental studies about PM toxicity and the related health impairments. For example, 95% rats developed dysphasia and carcinogenesis over a period of time of dermal pasting PM [23]. Further effects, including leukoplakia, submucous fibrosis, loss of nuclear polarity, keratosis, parakeratosis, inflammatory cell infiltration, vascularity and decreased weight of the gonads and the brain [24], were found. Bone marrow and chromosomal deformities in mice fed PM were also reported [25]. Recently, carcinogenic effects were confirmed in rats fed PM for 56 weeks [20]. Our data addresses the composition of PM and its po and ip effects, over both short-term and long-term periods. According to our data, the toxicity of PM administered po and ip in mice is obvious, and the toxicity depends on the constituents and the dose administered. The areca nut has significant toxicity [26]. It contains many alkaloids, and its active component, arecoline, has been shown to induce cancer as a result of its reaction with cysteine in vivo and in vitro. The reaction between polyphenols and catechu and lime produces cysteine/3 alkylation addicts and superoxide ions and antioxidants [27].

In practice, many forms of PM consumption and the increasing number of components in PM betel quid cause a wide range of health impairments. For instance, catechu and lime are smeared on the betel leaf, and then pieces of areca nut and tobacco are added in addition to flavoring agents (such as cardamom, cloves, coconut, ginger, sugar and gulkhand (rose petals and sugar)) before betel quid consumption. Thus, clinical assessment is difficult and must include tracing the etiology of disease by recording the history and lifestyle of the patient. The tobacco in betel quid (28) and catechu (tannin) in PM (IARC 1985) were found to cause cancer. Chewing areca nuts caused submucous fibrosis [29] but seldom ended in malignant cancer [30]. PM, BQ and areca nut chewing resulted in oral submucosis much earlier in a younger group that consumed only PM. Cytogenic anomalies, such as sister chromatid exchange, chromosomal aberrations and micronuclei were found in PM and tobacco consumers [8].