Hemoglobin adducts of the human bladder carcinogen o-toluidine after treatment with the local anesthetic prilocaine
Introduction
Prilocaine is a local anesthetic of the amide type and was introduced in 1959 (Wendl, 1965). It is widely used for local anesthesia either alone (e.g. Citanest®, Xylonest®) or in EMLA® (eutectic mixture of local anesthetics) products together with lidocaine (Buckley and Benfield, 1993, de Weert et al., 2000, Söderberg et al., 2006). In the tumescent technique for liposuction using large volumes (up to 6000 ml) of a 0.05% solution of lidocaine (Klein, 1987), it is recommended to replace lidocaine by prilocaine because of the lower toxicity of prilocaine for the cardiovascular and central nervous system (de Weert et al., 2000). Although the reported safe maximum dose of prilocaine in local anesthesia is 8.5 mg/kg body weight, up to 5-fold higher doses as used in tumescent local anesthesia do not lead to acute toxic side effects (Lindenblatt et al., 2004). Although prilocaine is a well-known methemoglobin (Met-Hb) inducer, Met-Hb did not increase over 1.4% and maximal plasma levels of prilocaine of only 0.34 μg/ml were well below the threshold of prilocaine CNS toxicity which is 5–6 μg/ml (Stymne and Lillieborg, 2001).
After amide ester hydrolysis lidocaine and prilocaine release 2,6-dimethylaniline and o-toluidine, respectively. In a previous study high hemoglobin adduct levels of 2,6-dimethylaniline have been detected in patients treated with lidocaine (Bryant et al., 1994). Since prilocaine releases o-toluidine to a considerable extent (Fig. 1), highly elevated hemoglobin adduct levels from o-toluidine are expected to result from treatment with prilocaine. Except 4-hydroxyprilocaine which has been identified only by co-chromatography on thin-layer plates as a minor metabolite in human urine, all other identified metabolites of prilocaine are secondary metabolites of o-toluidine (Åkerman et al., 1966, Hjelm et al., 1972, Son et al., 1980). In several studies o-toluidine has been detected in human plasma after administration of prilocaine or EMLA® products (Klein et al., 1994, van der Meer et al., 1999, Lok et al., 1999, Friskopp and Huledal, 2001, Herdevall et al., 2003).
o-Toluidine is not only responsible for Met-Hb formation observed after prilocaine treatment but also gives rise to DNA adducts (Fig. 1) and has a carcinogenic risk potential. It is classified by the International Agency for Research on Cancer (IARC) as probably carcinogenic for humans and is considered to be a proven human carcinogen by the German MAK Commission (IARC, 2000, Deutsche Forschungsgemeinschaft, 2006). Therefore, exposure to o-toluidine could have long-term toxicological consequences. Several epidemiological studies have suggested that occupational exposure to o-toluidine is associated with an increased risk of bladder cancer (Rubino et al., 1982, Ward et al., 1991, Markowitz and Levin, 2004, Markowitz, 2005). This has been corroborated by the detection of high adduct levels of o-toluidine-releasing hemoglobin adducts in workers of a rubber manufacturing plant (Ward et al., 1996). Generally, hemoglobin adducts are considered to be excellent surrogate markers of aromatic amine bioactivation to their ultimate carcinogenic metabolites (Skipper and Tannenbaum, 1990, Richter and Branner, 2002). For 4-aminobiphenyl, another human bladder carcinogen, a correlation between DNA adducts in exfoliated urothelial cells and hemoglobin adducts has been reported for smokers and nonsmokers (Talaska et al., 1991).
To verify the hypothesis that highly elevated hemoglobin adduct levels from o-toluidine may result from treatment with prilocaine, blood samples were analyzed before and 24 h after prilocaine treatment. Hemoglobin adducts of 4-aminobiphenyl were determined to control for smoking-related effects.
Section snippets
Participants
Overall, 29 subjects participated in the study, 23 patients undergoing surgery at the Department of Otorhinolaryngology of the Ludwig-Maximilians University of Munich, Germany, and 6 healthy volunteers from the Department of Otolaryngology of the University of Regensburg, Germany. All subjects were of Caucasian origin and had a mean age of 48 years (17–81) and a mean body weight of 73 kg (50–108). There were 21 men (9 smokers, 12 nonsmokers) and 8 women (2 smokers, 6 nonsmokers). Demographic
Results
o-Toluidine-releasing hemoglobin adducts were detectable in all individuals at baseline ranging from 0.037 to 40.908 ng/g hemoglobin. Excluding the female patient #9 with the highest adduct level, the basic adduct value was 0.54 ± 0.95 ng/g hemoglobin (Table 2). Treatment with 100 mg prilocaine resulted in a highly significant increase (P < 0.0001) of o-toluidine adducts by 21.48 ± 13.06 ng/g hemoglobin (Fig. 2). In patients #1, #4 and #23, receiving 50, 120 or 200 mg prilocaine, o-toluidine adducts
Discussion
It is for a long time known that o-toluidine is a primary metabolite of prilocaine (Hjelm et al., 1972, Åkerman et al., 1966). In patients receiving a mixture of lidocaine and prilocaine, o-toluidine was determined as a prilocaine metabolite in plasma (Klein et al., 1994, van der Meer et al., 1999, Lok et al., 1999, Friskopp and Huledal, 2001, Herdevall et al., 2003). However, the extent of metabolic activation of o-toluidine to proximal carcinogens has not been investigated. In the present
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