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Tramadol Is Prescribed

Tramadol is prescribed for the treatment of moderate to severe pains. It is considered as a centrally acting analgesic medicament (Musshoff and Madea, 2001). Mortality recorded due to overdose concentrations of tramadol has been reported in several studies (Lusthof and Zweipfenning, 1998; Mitchuad et al., 1999; Moore et al., 1999; Klingmann et al., 2000; Musshoff and Madea, 2001; Loughrey et al., 2003; Bynum et al., 2005; De Decker et al., 2008).

After death, the cadaver became infested with different dipteraous larvae. Entomological techniques are the most trustworthy to determine the minimum post mortem interval (PMI). PMI describes the time between the discovery of the corpus and its infestation by the insects (Bourel et al., 1999). Two ways could be used, the first is the pattern of the insect succession and the second is to use the insect growth rates (Bourel et al., 1999). Maggots collected from decomposed corpus, can serve as alternate samples reflecting the evidence or the absence of the drug in the body (Beyer et al., 1980; Wilson et al., 1993; Kintz et al., 1994; Sadler et al., 1995; Hedouin et al., 1999; De-Letter et al., 2000). In some instances where the whole cadaver tissues and fluids were completely decomposed, collected fly larvae can give clear evidence if the corpus was drug abused or not (Beyer et al., 1980; Kintz et al., 1990A). Analysis of the maggot tissues (Hedouin et al., 1999; Pounder, 1991; Introna et al., 1990) and puparia (Introna et al., 1996; Wilson et al.; 1993) using different techniques such as TLC or Liquid chromatoprapghy will determine the amount of the drugs present in the body of the maggots and also can serve to deduce or expect the amount of drugs accumulated within abused or overdosed corpuses (Kentz et al., 1990 B, 1990 C; Introna et al., 1990).

When certain toxic materials such as drugs are present within the corpse tissues, it can affect the larval growth rates, leading to inaccurate determination of the minimum PMI. Previous studies investigated the effect of the drugs on the growth rates of the blowflies, in case of morphine (George et al., 2009; Bourel et al. 1996 and 1999); paracetamole (O’Brien and Terner, 2004); codein (Kharbouche et al., 2008); tramadol (Lamia et al., 2011) and diazepam (Carvalho et al., 2001).

Little studies dealt with the effect of drugs on the developmental rates of the flesh flies, for example, in case of cocaine, Goff et al. (1989); Goff et al. (1991) in case of heroin; methamphetamine, (Goff et al., 1992); amitriptyline, (Goff et al., 1993); phencyclidine (Goff et al., 1994) and methenedioxymethamphetamine, (Goff et al., 1997).

The main goals of the present study are: 1- To investigate qualitatively and quantitatively the amount of tramadol or its metabolites consumed by the sarcophagid larvae and pupae from tramadol treated rat livers. 2- To determine the effect of accumulated tramadol inside rat livers on the duration and the development, of the larvae and pupae of certain sarcophagid flesh fly S. argyrostoma.

Materials and Methods

– Animal model:

Wistar albino strain Rattus norvegicus was used as the animal model during the progress of this study. With an average weight of 200 ± 10.9 g, 90 rats were used and were kept at the animal house of Zoology Department, under controlled temperature (25–27°C) and relative humidity (30–40%). The Committee of Zoology Department, Faculty of Science, Fayoum University, on Animal Research and Ethics (FU-CARE) permitted the use of the experimental rats. FU-CARE followed the European convention for the protection of vertebrate animal used for experimental and other scientific purposes (2005) (http://conventions.coe.int/Treaty/EN/Reports/HTML/123.htm) in addition to the guidelines for the accommodation and care of animals used for experimental and other scientific purposes (C(2007)2525: http://ec.europa.eu/transparency/regdoc/rep/3/2007/EN/3-2007- 2525-EN-1-0.Pdf).

– The stock colony of the flesh fly Sarcophaga argyrostoma

AbouZied (2016) recorded that adults and larvae of S. argyrostoma, were the most attracted and abundant species among fly fauna towards addicted rat carcasses, in the campus of Fayoum University. Therefore, the colony was established from a single wild female catch, since 2014. Genitalia of the third generation males were dissected under stereomicroscope, cleared in potash and examined by M. El-Hawagry (Professor of insect taxonomy, Entomology Department, Cairo University). Genitailia were also sent to Thomas Pape (Professor of Taxonomy, Danish Natural History Museum, Copenhagen) for justifying our identification. The colony was reared inside the insectary of Faculty of Science, Fayoum University. The laboratory temperature ranged from 23 – 26 ◦C and 45 – 50 % relative humidity. The photoperiod ranged from (16:8 L/D).

– Dose determination

Tramadol hydrochloride tablets (200 mg) (Zydol® SR Tabs; Grünenthal Ltd, UK), were used during the progress of this study. Each tablet (weight 0.35 ± 0.03 g) was ground in a dry-sterilized porcelain mortar. According to Paget and Barnes (1964), the corresponding dosage for the albino rats was calculated as equal to 6.3 mg/200 g rat. Two concentrations of tramadol were used, the recommended dose (D1) and an overdose (D2). In case of the recommended dose (D1), 6.3 mg of the tramadol powder was dissolved in 0.2 ml distilled water. In case of the over dose (D2), 12.6 mg was dissolved in 0.2 ml distilled water. Each rat was administered once a day, using stomach tube.

Rats were arranged into 3 groups (30 rats each). One group was injected with the recommended dose (D1). Another group of 30 rats was injected with the over dose (D2). The last group was injected with 0.9 saline solutions as the control. After 3 months of tramadol administration (n= 90 doses), rats were killed by cervical dislocation (Cressey 2013), then dissected in isotonic saline solution. Livers were removed from each rat, preserved in plastic bags, labeled and stored in freezer until using to nourish the flesh fly larvae. One gram from each liver was cut off by sharp knife blade, and then labeled either as control, (D1) liver or (D2) liver, frozen at (-20oC) for extraction and further GCMs analysis.

-Reagents and chemicals

Tert-butyl methyl ether, n-hexane, acetone (HPLC grade solvents) were purchased from Redel and Fluka (Germany). Solvents were used during all procedures without further purification. From Sigma-Aldrich (Germany), borax was obtained. By means of a Milli-Q water purification system (Millipore, Bedford, MA, USA), high purity water was obtained and used in all procedures.

– Sample preparation

Half gram of each sample (rat liver, larvae and pupae) was accurately weighed and transferred to glass beaker. Borax (0.5 ml) was added to adjust pH at 9. The suspension was homogenized and mixed well with a glass rod, and allowed to stand for 30 minutes. Two ml of tert-butyl methyl ether was added. The test tube was firmly capped, shaken vigorously for 5 minutes and the tube was left for 10 minutes. For phase separation, the upper phase was filtered using fine syringe filter (0.2 µm) and the filtrate was transferred to GC vials for analysis.

– Gas chromatography

The chromatographic analysis of tramadol was carried out using gas chromatography Agilent 6890N equipped with a 7683B automated injector, flame ionization detector (FID) and 5975 inert XL mass selective detector. The chromatographic separation was achieved with a non polar column, DB-5MS (60 m, ID 0.25 mm and film thickness 0.25 µm) from JW Scientific (USA). The carrier gas used was helium at constant flow rate of 1.0 ml/min. Split less injection mode was used and the injection performed at 260 oC. The oven temperature was programmed as follows: initial temperature (100 oC), then increased by 10oC/min to 310 oC and held isothermal for 20 minutes at this temperature. The mass spectrometer was scanned from m/z 50 to 500. The ion source, quadruple and interface temperatures were 230, 150 and 310 oC, respectively. Agilent Chemstation Rev., B.02.01-SR1 (260) and MSD Chemstation D.02.00.275 was used to analyze the chromatographic data. The tramadol and its metabolites, if present, was identified by their retention times and confirmation of identity was performed by mass selective spectrometer (GC-MS).

-Estimation of the tramadol consumed by larvae of S. argyrostoma:

One day after female larviposition, 3 groups of larvae (60 larvae each) were picked up under stereomicroscope. Each group was supplied with 60 gm of the corresponding liver. One group fed on liver free tramadol (control), the second group fed on (D1) livers, and the third one fed on (D2) liver. After 3 days, 10 larvae from each group were killed with boiling water, weighted, labeled, and stored at (-20oC) in a polystyrene test tubes. The remaining alive larvae (n=50) were reared till pupation. One week after pupation, 10 pupae from each group were picked up by fine forceps, weighted individually from each group, and then stored as described in case of the larvae. The stored samples of the livers, larvae and pupae were sent for the laboratory of the National Institute of Standards, for further tramadol extraction and chromatographic GCMs analysis.

-Effect of the accumulated tramadol doses in rat livers, on the developmental stages of the flesh fly Sarcophaga argyrostoma

The remaining alive 50 larvae from each group were kept in plastic containers (50 cm diameter and 30 cm depth) half filled with moistened fine saw dust and covered with muslin and fastened with rubber band. Containers were labeled with date and the case of study. During the 4th day, 30 larvae were picked up randomly from each group; weighted individually using Analytical Sartorius Cubis series balance (max. 250 g, 0.00001g range). After weighting, larvae were returned back again to its’ original group. Containers were examined daily to record, the mortality of the larvae and the date of the development into pupae. Once pupation was established, they were kept in Wooden rearing adult fly boxes. One week after pupation, all the pupae were weighted individually. Boxes were supplied with powder sugar, and glass bottle filled with moistened cotton piece as a water source for future emerging adults. The laboratory temperature ranged from 23- 26 ◦C and 45- 50 % relative humidity. The photoperiod ranged from (16:8 L/D).

– Statistical analyses

Complex online statistical calculator was used to compare between the mean values of the weights and durations of both, the larvae and the pupae of the three cases (the control, D1 and D2 fed larvae). One way ANOVA followed with post-hoc Tukey HSD was established using test calculator which is freely available at http://astatsa.com/OneWay_Anova_with_TukeyHSD/

Results

Identification and quantitation of tramadol

At 21.08 minutes, a characteristic peak appeared in the chromatogram of treated liver samples (Fig. 1 A). Compared with the chart of the control, no diagnostic peaks were found, reflecting the complete absence of any tramadol traces (Fig. 2 A). Mass spectrum of tramadol treated liver samples detected a compound which has a peak corresponding to its molecular weight at m/z 263 in addition to ion base peak at m/z 58 (Fig. 3). For identification and quantitation purposes, the GC–MS analyses were performed in Scan/SIM mode at two ions 58 and 263 m/z and the typical retention time 21.08 minutes (Fig. 1 b and 2 b). All these criteria are unique for tramadol.

Quantitative analysis revealed that larvae of S. argyrostoma fed on D1 liver ingested lower concentration of tramadol (0.11 mg/g) compared to that content ingested from D2 liver (0.16 mg/g). Meanwhile, the amount of the tramadol persisted in pupae tissues were 0.07 and 0.09 mg/g, respectively, in cases of D1 and D2 liver. The accumulated tramadol concentrations in the livers of the two cases, D1 (0.72 mg/g) and D2 (1.62 mg/g) were higher than that detected in larvae and pupae samples (Table 1).

-The effect of tramadol on the development of the larval stages of the flesh fly Sarcophaga argyrostoma (Robineau-Desvoidy, 1830):

Out of 50 larvae used, only 32 larvae were alive after one day feeding on (D2) treated rat liver (mortality rate 36%). Meanwhile, larvae fed on (D1) treated liver tissue suffered lower mortality rates as 4% compared to 2% , in case of that of the control (Table 1).

The larvae that fed on (D2) rat livers showed lower duration (6.34 ± 0.48 days) compared to (7.04 ± 1.02 days; F= 4.1586; p=0.0001771) in case of the control. The larvae fed on (D1) treated liver showed also significantly shorter duration, compared to the control (6.64 ± 0.49 days, F= 2.6732; p = 0.0255466). Statistically, no significant difference (F= 1.7426; p=0. 2515) was detected between the change in the durations of the larvae fed on (D1) and (D2) treated livers (Table 2).

During the 4th day, larvae of the control were significantly larger, weighting (16.08 ± 1.49 mg) compared to the average weight of the larvae fed on D2 liver (13.43 ± 3.06 mg, F=5.3772, p=3.7045e-6). Also, the weight of the control larvae was significantly larger than that of the weight of the larvae fed on D1 livers (13.10 ± 1.28; F= 5.6675, p=987e-7).

-The effect of tramadol on the development of the pupal stages of the flesh fly S. argyrostoma (Robineau-Desvoidy, 1830):

Statistical analysis revealed that the pupae produced from larvae fed on (D1) treated liver acquired, significantly, longer duration (14.96 ± 0.66 days) compared to both, the case of the control (13.92±1.01 days; F= 6.5179; p= 4.8 e-9), and the case of the pupae from larvae fed on (D2) treated liver (14.38 ± 0.49 days; F= 3.2660; p= 0.0042) (Table 2).

Pupae produced from larvae which fed on (D2) treated livers acquired significantly (F= 3.1404; p=0.006) higher weight (15.91 ± 0.29 mg) compared to pupae produced from larvae fed on the control liver (14.18 ± 0.47 mg). Meanwhile the pupae produced from larvae fed on (D1) treated liver showed insignificant (F=2.1600; p=0.082) higher weight (15.24 ± 0.30 mg), compared to that fed on the control liver during the larval stage. Pupae resulted from the larvae fed on the liver treated with (D1) and the corresponding liver treated with (D2) showed insignificant change in weight (F= 1.2518; p= 0.427) (Table 2).

DISCUSSION

Entomotoxicology is defined as the analysis of toxins within the tissues of carrion feeding insects such as flies and beetles. Chromatograms revealed the presence of the tramadol inside the treated liver tissues (D1 and D2) of the rat. Tramadol metabolites were completely absent. The GC-Ms of the control liver tissues was free from both the tramadol and its metabolites. Similar results were obtained by Lamia et al. (2011). The authors detected tramadol by (HPLC) in various organs of experimentally injected rabbits, including the liver. In contrast, Budd and Langford (1999) concluded that tramadol is rapidly metabolized to O- and N- desmethyl tramadol, in the human liver. Additionally, about 10-30 % of the tramadol dose is excreted unchanged in the urine.

Chromatograms and mass spectrometry revealed that larvae and pupae of S. argyrostoma contained the tramadol. A possible explanation for the appearance of the tramadol is that while feeding, larvae of S. argyrostoma ingested the liver tissues together with the incubated tramadol. The tramadol persisted until the larvae reached pupation, but at very low concentrations (0.07 and 0.09 mg/g). Lamia et al. (2011) detected tramadol within tissues of the larvae of Lucilia sericata (Meigen). In comparison with other drugs, Beyers et al. (1980) analyzed the larvae of Cochliomyia macellaria (Fabricius) (Calliphoridae) collected from remains of the cadaver tissue. Analysis of C. macellaria larvae tissues revealed the presence of phenol barbital, 14 days after female cadaver suicide, when no tissues or fluids were available. Kintz et al. (1990A) found that calliphorid larvae contained 5 drugs (triazolam, oxazepam, phenol barbital and clomipramine) two months after corpus death. Morphine and phenol barbital were both detected in calliphorids larvae developed on chronic heroin abused cadaver (Kintz et al., 1990B). Physiologically, when the rate of drug absorption exceeds the rate of drug elimination by both the Malpighian tubules and the nephrocytes, the drugs appeared in maggot tissues (Chapman, 1928).

In this study, tramadol detected in the tissues of S. argyrostoma larvae was lower (6.5-9 times) compared to the tramadol content in D1 and D2 rat livers. Hedouin et al. (1999) found that drugs in larvae were 30-100 times lower than that in tissues of the cadavers. The same was true in case of maggots of Calliphora vicina (R-D), fed on overdosed cadaver tissues with proxamol and amitriptyline (Wilson et al., 1993). Tracqui et al. (2004) and Campobasso et al. (2004) reported that the concentrations of the drugs present in the larvae were much lower than their concentrations in the cadavers. Kintz et al. (1990C) stated that there was a correlation between drugs in human tissues and the amount of drugs detected in maggots fed on such tissues. Introna et al. (1990) recorded a significant correlation (r=0.79) between the concentrations of opiates found in larvae and that in the liver tissues. However, Pounder (1991) and Hedouin et al. (1999) didn’t record any correlation between drug concentration in maggots and that in the cadaver tissue.

Pupae of S. argyrostoma contained a very low concentration of tramadol as 0.07 and 0.09 mg/g, respectively, when compared with 0.72mg/g (D1) and 1.62 mg/g (D2). Introna et al. (1996) recorded morphine from empty puparia of C. vicina, which fed on morphine mixed substrates during the larval stage. However, in case of puparia and adults of C. vicina which were fed during their larval stage on overdosed co-proxam and amitriptyline, both were free from any drugs (Wilson et al., 1993).

Previous studies demonstrated that the presence of drugs and toxins can alter the developmental rates of carrion insects feeding on decomposed tissues of the cadavers (Catts and Goff, 1992; Goff et al., 1992; Bourel et al., 1999; Goff and Lord, 1994; Byrd and Castner, 2000; O’brien and Turner, 2004). In this study, the presence of the tramadol in the liver tissues of treated rats (D1 and D2), was the reason for decreasing the duration of the larvae of S. argyrostoma, but increased the duration of the pupal stage. AbouZied (2016) found that larvae of S. argyrostoma fed on tramadol treated rat carcasses pupated two days after the pupation of the control larvae. Murthy and Mohanty (2010) concluded that heroin speeded up the larval growth of the carrion insects and then decreased the development rate of the pupal stage. Verma and Paul (2013) stated that cocaine and methamphetamine accelerated the rate of the development of the flesh fly Parasarcophaga ruficornis (Fabricius) (Diptera: Sarcophagidae).

The effect of the toxins on the arthropods depends on the concentration of the toxin (Murthy and Mohanty, 2010). Therefore, the over dose (D2) caused a larval mortality rate of 36%, followed by 4% in the case of (D1) concentration. Goff et al. (1993) found that the larval mortality rates of P. ruficornis were significantly greater, in colonies fed on amitriptyline liver than the mortality rate of the control colony. In addition, the mortality rates of the treated larvae were inversely correlated with the concentrations of the drug (amitriptyline) in the liver (Goff et al., 1993).

Larvae fed on (D2) and (D1) liver fed recorded short longevity compared to larvae fed on control liver. Similarly, the lethal dose of cocaine caused larvae to develop rapidly, 36 – 76 hours after hatching (Gagliano-Candela and Aventaggiato, 2001).

Pupae resulted from larvae fed on D1 and D2 acquired longer durations compared to that corresponding case of the control. Similarly, colonies of Boettcherisca peregrina (Robineau- Desvoidy) fed on tissues of heroin dosed rabbit, required longer pupation time, when compared with that of the control colony (Goff et al., 1991). Additionally, with 600- 1000 mg of amitriptyline treated livers, pupae of P. ruficornis required a significantly longer duration, compared to the control colony (Goff et al., 1993). Pupation occurred earlier in larvae of B. peregrina (Sarcophagidae) feeding on tissues with higher concentrations of cocaine, benzoylecognine, or both (Goff et al., 1989). However, in case of cocaine, pupal duration showed insignificant difference among colonies of B. peregrina related to the concentrations of the cocaine or its metabolite benzoylecognine in tissues (Goff et al., 1989). In contrast, pupation occurred earlier when feeding on tissues containing higher concentrations of heroin (Goff et al., 1991).

Pupae produced from (D2) liver fed larvae had a higher weight compared to that of the control fed larvae and (D1) fed larvae. Pupae from D1 and the control showed similar weights. Similarly, the lethal and twice-lethal dosages of heroin resulted in larger maggots of B. peregrine (Sarcophagidae) in all the treated colonies until the maximum size was attained (Goff et al., 1998). Also, the sarcophagid larvae B. peregrina fed on the control and that fed on the sub lethal dose of cocaine showed nearly the same development rate (Goff et al., 1998). Morphine and heroin were both believed to slow down the rate of fly development (Introna et al., 2001). Conclusion

Data of the present study suggested that larvae of S. argyrostoma could be used as taxa to determine the minimum PMI taking into consideration the decrease of the larval duration, in Egypt. Larvae and pupae of S. argyrostoma can give brief evidence about the presence or absence of any drugs, especially, when the tissues and body fluids of the cadavers are completely consumed.