Skeletal stability after mandibular advancement in bilateral sagittal split osteotomies during adolescence.

Bilateral sagittal split osteotomy (BSSO) is the most frequently performed surgery for correcting mandibular retrognathia. Few studies have reported the use of BSSO in young patients, as growth may cause relapse. The aim of the present study was to determine the amount of relapse after performing BSSO in patients aged less than 18 years. Patients who had a mandibular advancement by BSSO surgery between January 2003 and June 2008 were evaluated. Eighteen patients were treated before the age of 18 years and compared with patients treated at 20-24 years of age. Cephalometric radiographs were used to determine the amount of relapse. For patients aged less than 18 years, the mean horizontal relapse after 1 year was 0.5 mm, (10.9% of perioperative advancement). For patients aged 20-24 years, the mean relapse was 0.9 mm, (16.4% of perioperative advancement). There were no significant differences between the age groups (p > 0.05). In conclusion, the BSSO procedure is a relatively stable procedure, even during adolescence.

Comparative biotransformation of hexachlorobenzene and hexafluorobenzene in relation to the induction of porphyria.

The porphyrinogenic action of hexafluorobenzene was investigated and compared to that of hexachlorobenzene. Metabolite patterns in the urine of exposed rats were determined to quantify the extent of metabolism through cytochrome P450 catalysed oxidation and glutathione conjugation. Results obtained demonstrate an almost similar extent of formation of phenolic metabolites. However, in the urine of hexachlorobenzene exposed rats significantly higher levels of the N-acetyl-S-(pentahalophenyl)cysteine were observed than in the urine of hexafluorobenzene exposed rats. Hexafluorobenzene exposure did not result in induction of porphyria, whereas exposure to hexachlorobenzene did result in significantly elevated levels of urinary as well as liver porphyrins. Together these results indicate that if the reactive intermediate is indeed formed in the cytochrome P450 catalysed initial oxidative dehalogenation, the extent of its formation as well as its subsequent reactivity and reaction pathways vary with the type of the halogen substituents. Furthermore, the results seem to indicate that the extent of metabolism of hexahalogenated benzenes into urinary metabolites resulting from glutathione conjugation is a better indication of their porphyrinogenic action than their extent of metabolism to phenolic metabolites. Two explanations for this observation are presented.

Immune effects of hexachlorobenzene in the rat: role of metabolism in a 13-week feeding study.

The role of metabolism and porphyria in the immunomodulating effects of hexachlorobenzene (HCB) was investigated. To this end, female Wistar rats received a diet with two different doses of HCB and pentachlorobenzene (PCB), either in combination or not with the cytochrome P450IIIA1 inhibitor, triacetyloleandomycin (TAO). Urinary metabolites and urinary and liver porphyrins were measured at regular intervals and data have been published elsewhere. Skin lesions were scored weekly and after 13 weeks of exposure lymphoid organs were weighed, spleens were examined by morphometry, and serum.(auto)antibody levels were determined by ELISA. The probability of causal relationships between the different parameters was determined by analysis of correlation. HCB caused a dose-dependent increase of the incidence, but not the severity, of skin lesions, and dose-dependently increased weights of popliteal lymph nodes and spleen and serum levels of IgM, IgA, and autoantigen-specific IgM. IgG and IgG autoantibody levels were not changed. The splenomegaly could be attributed to an expansion of all splenic compartments. PCB caused no skin lesions and had only minor, predominantly immunosuppressive effects. TAO virtually lacked immunomodulating activity of its own, hardly affected the induction of skin lesions by HCB, did not change the immune effects of HCB, and suppressed IgG levels when combined with PCB. Comparison of the immunological data with those found in the same rats as to induction of porphyria and biotransformation of HCB and PCB, indicates that the HCB-induced porphyria, being markedly reduced by coadministration of TAO, is not involved in the immunomodulating effects of HCB. The same conclusion could be drawn for the oxidative HCB metabolites, since TAO inhibited their formation, while the same metabolites were formed upon administration of PCB that lacked the immunostimulatory effects of HCB. Therefore, HCB itself, its nonoxidative metabolites, or their precursors are likely candidates for inducing the immune effects. Further, an immune component in the HCB-induced skin lesions, usually associated with dermal porphyrin accumulation, is suggested by the observations that TAO profoundly reduced induction of porphyria, but not of skin lesions and immune effects, by HCB.

Biotransformation and toxicity of halogenated benzenes.

1. Multiple potentially harmful metabolites can be distinguished in the metabolic activation of halogenated benzenes: epoxides, phenols, benzoquinones and benzoquinone-derived glutathione conjugates. 2. The role of these (re-) active metabolites in the toxic effects induced by halogenated benzenes such as hepatotoxicity, nephrotoxicity, porphyria and thyroid toxicity is discussed. 3. Evidence is presented suggesting that the formation of reactive benzoquinone metabolites rather than the traditional epoxides is linked to halogenated benzene-induced hepatotoxicity. 4. A crucial role for the benzoquinone-derived glutathione adducts in halogenated benzene-induced nephrotoxicity is clearly established. 5. Although metabolic activation appears to be involved in porphyria, the nature of the ultimate porphyrinogenic metabolite has not been elucidated yet. 6. Disturbances in thyroid hormone (and retinoid) homeostasis can be (at least partially) explained by the formation of halogenated phenol metabolites. 7. In conclusion, for a relevant prediction of the ultimate fate of a compound in a living organism, one should know the chemical characteristics and reactivity of the parent compound and its metabolites, together with insight into the formation mechanism of each of the suspected metabolites, and an understanding of the interaction between a specific chemical (reactive) structure and its target molecule.

Comparison of the urinary metabolite profiles of hexachlorobenzene and pentachlorobenzene in the rat.

The urinary metabolite profile of hexachlorobenzene (HCB) and pentachlorobenzene (PCBz) in the rat is compared after dietary exposure for 13 weeks. Both HCB and PCBz are oxidized to pentachlorophenol (PCP) and tetrachlorohydroquinone (TCHQ), which were the only two mutual metabolites formed. Additional urinary metabolites of HCB are N-acetyl-S(pentachlorophenyl)cysteine (PCTP-NAC), which appeared to be quantitatively the most important product, and mercaptotetrachlorothioanisole (MTCTA), which was excreted as a glucuronide. PCBz is more extensively metabolized to the major metabolites 2,3,4,5-tetrachlorophenol (TCP), mercaptotetrachlorophenol (MTCP) and the glucuronide of pentachlorothiophenol (PCTP), and the minor metabolites methylthiotetrachlorophenol (MeTTCP), hydroxytetrachlorophenyl sulphoxide (HTCPS), and bis(methylthio)-trichlorophenol (bis-MeTTriCP). The biotransformation of HCB and PCBz was modulated by selective inhibition of cytochrome P450IIIA in rats which received combined treatment of HCB or PCBz with triacetyloleandomycin (TAO). Rats receiving this diet had a strongly diminished excretion of both PCP and TCHQ, as compared to rats fed HCB or PCBz alone, indicating the involvement of P450IIIA in the oxidation of both compounds. However, the excretion of 2,3,4,5-TCP was not diminished by co-treatment of rats with PCBz and TAO, indicating that: (i) the oxidation of PCBz to PCP and 2,3,4,5-TCP does not proceed via a common intermediate; and (ii) oxidation of PCBz to 2,3,4,5-TCP is not mediated by P450IIIA. Co-treatment of rats with PCBz and TAO had a differential effect on the excretion of sulphur-containing metabolites, resulting in a decrease in the excretion of PCTP glucuronide, whereas no change was observed in the excretion of MTCP, as compared to rats receiving PCBz alone. The observed differences in HCB and PCBz metabolites clearly deserve further in vitro studies to elucidate their origin.

Cytochrome P450-mediated oxidation of pentafluorophenol to tetrafluorobenzoquinone as the primary reaction product.

In the present study the oxidative dehalogenation of a para-halogenated phenol was studied using pentafluorophenol and its non-para-halogenated analogue 2,3,5,6-tetrafluorophenol as model compounds. 19F NMR was used to characterize the metabolite patterns. In order to study the primary oxidation products of the microsomal cytochrome P450-catalyzed conversion, the alternative oxygen donors cumene hydroperoxide (CumOOH) and iodosobenzene (IOB) were used in addition to the use of NADPH and molecular oxygen. In a NADPH/oxygen-driven reaction, but also in a CumOOH- or IOB-driven cytochrome P450 reaction, tetrafluorophenol was converted to tetrafluorohydroquinone. However, for pentafluorophenol, the formation of tetrafluorohydroquinone as a product of its cytochrome P450-mediated conversion was only observed in the NADPH-driven system. Addition of reducing equivalents such as NADH to the CumOOH or IOB incubations resulted in the formation of tetrafluorohydroquinone. From these data it was concluded that the primary reaction product of the cytochrome P450-catalyzed conversion of pentafluorophenol is a reactive species that can be reduced to tetrafluorohydroquinone by NAD(P)H and, thus, must be tetrafluorobenzoquinone. Additional experiments with tetrafluorobenzoquinone, incubated in vitro with either microsomal protein or glutathione in the presence or absence of reducing equivalents, demonstrated that the tetrafluorobenzoquinone ends up bound to proteins, losing its fluorine atoms as fluoride anions. Thus, while cytochrome P450-mediated conversion of the 2,3,5,6-tetrafluorophenol results in the formation of tetrafluorohydroquinone as the primary reaction product, monooxygenation at a fluorinated para position, such as in pentafluorophenol, results in the formation of the reactive tetrafluorobenzoquinone derivative as the primary reaction product.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of oxidative metabolism in hexachlorobenzene-induced porphyria and thyroid hormone homeostasis: a comparison with pentachlorobenzene in a 13-week feeding study.

Hexachlorobenzene (HCB) induces a broad spectrum of effects including disturbances in the heme synthesis (porphyria) and in thyroid hormone homeostasis. For most of its effects, biotransformation of the parent compound seems to be a prerequisite. The present study was designed to assess the relevance of the oxidative metabolites in HCB-induced toxicity, with special attention to the role of the reactive tetrachlorobenzoquinone (TCBQ). To this end, toxicity and biotransformation of HCB were compared with those of pentachlorobenzene (PCB), since this chemical is oxidized to the same products as HCB, i.e., pentachlorophenol (PCP) and TCBQ. Female Wistar rats received diets containing different dose levels of HCB or PCB for 13 weeks, with or without cotreatment with triacetyloleandomycin (TAO), a selective inhibitor of cytochrome P450IIIA1/2. Rats treated with HCB (high dose) had significantly elevated levels of urinary porphyrins from the 4th week on and had a significant hepatic accumulation of porphyrins at the end of the study. Both urinary porphyrin excretion and hepatic porphyrin accumulation were greatly inhibited in rats receiving cotreatment with HCB and TAO. However, the inhibition of HCB-induced porphyria by TAO cannot be explained by a diminished formation of the highly reactive TCBQ, since rats treated with a high dose of PCB, which had a several fold higher urinary excretion of PCP and TCHQ compared to a high dose of HCB, did not develop porphyria. Instead, the present study points to the involvement of a putative reactive intermediate in the primary oxidative step in HCB-induced porphyria, since based on paired observations of individual rats, the degree of porphyria was correlated to a high degree with excretion of PCP, whereas correlation of porphyria with early excretion of TCHQ was much weaker. This finding fits well with the fact that the mechanisms of oxidation of HCB to PCP and PCB to PCP are different. Both HCB and PCB were oxidized to PCP and tetrachlorohydroquinone (TCHQ), the reduced analog of TCBQ. Cytochrome P450IIIA1/2 appears to be involved in the conversion of HCB and PCB, since cotreatment of TAO resulted in a strongly diminished urinary excretion of PCP and TCHQ. Treatment with HCB as well as PCB results in disturbances of retinoid and thyroid hormone homeostasis. These effects, which have also been reported after exposure to polychlorinated biphenyls, originate from interference of hydroxylated metabolites (notably PCP) with the plasma thyroxine transport protein, transthyretine, and since this metabolite is formed from both HCB and PCB, this results in the same toxicity for both compounds.

The involvement of primary and secondary metabolism in the covalent binding of 1,2- and 1,4-dichlorobenzenes.

The microsomal oxidation of 1,2-[14C]- and 1,4-[14C]dichlorobenzene (DICB) was investigated with special attention for possible differences in biotransformation that might contribute to the isomer-specific hepatotoxicity. Major metabolites of both isomers were dichlorophenols (2,5-DICP for 1,4-DICB and 2,3- and 3,4-DICP for 1,2-DICB, respectively) and dichlorohydroquinones. The formation of polar dihydrodiols appeared to be a major route for 1,2-DICB but not 1,4-DICB. Both the hepatotoxic 1,2-DICB and the non-hepatotoxic 1,4-DICB were oxidized to metabolites that covalently interacted with protein and only to a small extent with DNA. Protein binding could be inhibited by the addition of the reducing agent ascorbic acid with a concomitant increase in the formation of hydroquinones and catechols, indicating the involvement of reactive benzoquinone metabolites in protein binding. However, in the presence of ascorbic acid, a substantial amount of protein-bound metabolites of 1,2-DICB was still observed, in contrast to 1,4-DICB where binding was nearly completely inhibited. This latter effect was ascribed to the direct formation of reactive benzoquinone metabolites in a single P450-mediated oxidation of para-substituted dichlorophenols (such as 3,4-DICP) in the case of 1,2-DICB. In contrast, the major phenol isomer derived from 1,4-DICB (i.e. 2,5-DICP) is oxidized to its hydroquinone derivative, which needs prior oxidation in order to generate the reactive benzoquinone species. Residual protein binding in the presence of ascorbic acid could also indicate the involvement of reactive arene oxides in the protein binding of 1,2-DICB, but not of 1,4-DICB. However, MO computer calculations did not provide indications for differences in chemical reactivity and/or stability of the various arene oxide/oxepin tautomers that can be formed from either 1,2-DICB or 1,4-DICB. In conclusion, reactive intermediates in the secondary metabolism of 1,2-DICB lead to more covalent binding than those derived from 1,4-DICB, which correlates very well with their reported hepatotoxic potency.

The liver, kidney, and thyroid toxicity of chlorinated benzenes.

The acute toxicity of a number of chlorinated benzenes, ranging from monosubstituted to pentasubstituted benzenes, was studied in rats. Toxic effects on the liver, the kidneys, and the thyroid were monitored after a single ip administration of 1, 2, or 4 mmol/kg monochlorobenzene (MCB), 1,2-dichlorobenzene (1,2-DICB), 1,4-dichlorobenzene (1,4-DICB), 1,2,4-trichlorobenzene (1,2,4-TRCB), and pentachlorobenzene (PECB). Due to its low solubility, 1,2,4,5-tetrachlorobenzene (1,2,4,5-TECB) was tested at a highest dose of 0.8 mmol/kg. 1,2-DICB and 1,2,4-TRCB produced the most severe hepatotoxic effects when compared with an equimolar dose of the other chlorinated benzenes, as determined by plasma ALT profile and histopathological changes after 72 hr. MCB was considerably less hepatotoxic. Severe degenerative damage to the kidney was only observed in a few rats treated with 1,2,4-TRCB. However, protein droplets in the tubular epithelial cells were observed at 72 hr after administration of 1,4-DICB, 1,2,4-TRCB, 1,2,4,5-TECB, and PECB. In the latter two groups, these protein droplets were still observed 9 days after administration. All chlorinated benzenes tested excluding MCB induced a reduction in plasma thyroxine levels. The extent of decrease in plasma thyroxine was more severe in rats treated with 1,2,4-TRCB or PECB and correlated well with the relative binding affinities of the phenolic metabolites to the plasma transport protein for thyroxine, i.e., transthyretin. The present study indicates that the establishment of a structure-activity relationship with regard to toxicity depends on the sensitivity of the respective target organs. In the series of (poly)chlorinated benzenes studied, ranging from mono- to pentachlorobenzene, the most severe effects on liver, kidney, and thyroid were observed for 1,2,4-substitution.

Metabolic activation of 1,2,4-trichlorobenzene and pentachlorobenzene by rat liver microsomes: a major role for quinone metabolites.

Microsomal metabolism of 1,2,4-[14C]trichlorobenzene (1,2,4-TrCB) and [14C]pentachlorobenzene (PeCB) was studied with special emphasis on the conversion-dependent covalent binding to protein and DNA. 1,2,4-TrCB was metabolized to 2,3,6- and 2,4,5-trichlorophenol, and to a lesser extent to 2,4,6- and 2,3,5-trichlorophenol, and trichlorohydroquinone. About 10% of all metabolites became covalently bound to protein in a rather nonselective way. For 1,2,4-TrCB and PeCB a strong correlation between secondary metabolism to hydroquinones and covalent binding was established. Protein binding was completely inhibited by the addition of ascorbic acid, indicating quinone metabolites as the sole reactive species formed. Both 1,2,4-TrCB and PeCB alkylated DNA, although to a much lesser extent than protein (0.5 and 0.3% of all metabolites, respectively). Nonquinone intermediates, presumably epoxides, were responsible for a minor portion of the observed DNA binding, since complete inhibition by ascorbic acid was not reached. The differential role of cytochrome P450 both in primary and in secondary metabolism was demonstrated by the use of microsomes from rats pretreated with different inducers. Dexamethasone (DEX) microsomes (cytochrome P450IIIA1) showed the highest activity toward these chlorinated benzenes (14 nmol/mg/5 min for 1,2,4-TrCB and 36 nmol/mg/10 min for PeCB, both with regard to the formation of phenols and to the formation of protein-bound metabolites. In addition, DEX microsomes preferentially formed 2,3,6-trichlorophenol, whereas other microsomal suspensions formed 2,4,5-trichlorophenol as the major isomer. The present study clearly demonstrates the high alkylating potency of secondary quinone metabolites derived from chlorinated benzenes and poses a need for reevaluation of the role of epoxides in the observed toxicity of these compounds.

Studies on the mechanism of coumarin-induced toxicity in rat hepatocytes.

Rat hepatocyte suspensions have been used as a model system for some studies on the mechanism of coumarin-induced hepatotoxicity. Hepatocytes were isolated from male Sprague-Dawley rats and subjected to Percoll centrifugation to obtain preparations with >/=92% viability. Coumarin produced time- and concentration-dependent cytotoxic effects in rat hepatocytes as indicated by loss of cell viability and glutathione depletion. [3-(14)C]Coumarin was metabolized by rat hepatocytes to polar metabolites including o-hydroxyphenylacetic acid and to metabolites that became covalently bound to hepatocyte proteins. The addition of 10 mum-ellipticine significantly reduced coumarin cytotoxicity, coumarin metabolism and covalent binding in rat hepatocytes. These results demonstrate that coumarin-induced liver injury in the rat can be modelled in hepatocyte suspensions and that toxicity appears to be due to one or more cytochrome P-450 generated metabolites.

The metabolism of pentachlorobenzene by rat liver microsomes: the nature of the reactive intermediates formed.

Metabolism of [14C]-pentachlorobenzene by liver microsomes from dexamethasone-induced rats results in the formation of pentachlorophenol and 2,3,4,6-tetrachlorophenol as major primary metabolites in a ratio of 4:1, with 2,3,4,5- and 2,3,5,6-tetrachlorophenols as minor metabolites. The unsubstituted carbon atom is thus the favourite site of oxidative attack, but the chlorine substituted positions still play a sizable role. As secondary metabolites both para- and ortho-tetrachlorohydroquinone are formed (1.4 and 0.9% of total metabolites respectively). During this cytochrome P450-dependent conversion of pentachlorobenzene, 5-15% of the total amount of metabolites becomes covalently bound to microsomal protein. Ascorbic acid inhibits this binding to a considerable extent, indicating that quinone metabolites play an important role in the binding. However, complete inhibition was never reached by ascorbic acid, nor by glutathione, suggesting that other reactive intermediates, presumably epoxides, are also responsible for covalent binding.

Active site-directed irreversible inhibition of glutathione S-transferases by the glutathione conjugate of tetrachloro-1,4-benzoquinone.

Purified glutathione S-transferase from rat liver cytosol are irreversibly inhibited by the glutathione conjugate of tetrachloro-1,4-benzoquinone, 2-S-glutathionyl-3,5,6-trichloro-1,4-benzoquinone. The inhibition is due to covalent binding in or near the active site, resulting in modification of a single amino acid residue/subunit, presumably a cysteine residue. The amount of inhibition is related to the molar ratio of the inhibitor and the enzyme and is independent of the enzyme concentration. A 70-80% inhibition is obtained on incubating the enzyme with a 5-fold molar excess of the conjugate. Complete 100% inhibition is never reached. The derivative bound to the enzyme still possesses a quinone structure and is able to react with thiol-containing compounds. Reduction of the enzyme-bound quinone abolishes its reactivity but does not decrease the inhibition. At 0 degrees C, the glutathione conjugate of tetrachloro-1,4-benzoquinone inhibits the glutathione S-transferases at a much higher rate than the corresponding beta-mercaptoethanol conjugate, indicating a distinct targetting effect of the glutathione moiety. However, the parent compound, tetrachloro-1,4-benzoquinone, also has a considerable affinity for the enzymes. Although it does not react as fast as the glutathione conjugate, it reacts with the same amino acid residue. Protection from inhibition by the substrate analog S-hexylglutathione also indicates an active site-directed modification. Small but significant differences exist between the different rat liver transferase isoenzymes; using a 20-fold molar excess the inhibition ranges from 78 to 98% for the conjugate, and from 72 to 93% for the quinone, with isoenzyme 1-1 being the most and isoenzyme 2-2 the least inhibited forms.

Resting metabolic rate and diet-induced thermogenesis in abdominal and gluteal-femoral obese women before and after weight reduction.

Fifteen premenopausal obese women, seven abdominal obese (AO) and eight gluteal-femoral obese (GFO), followed an energy-reduced diet of 1000 kcal/d (4.2 MJ/d) over 8 wk. Body-fat distribution was assessed using a cutoff point of 0.80 for the waist-to-hips girth ratio. Before and after the dietary treatment resting metabolic rate (RMR) and diet-induced thermogenesis (DIT) (after a normal breakfast) were measured by indirect calorimetry. Body-weight reduction and energy intake during the diet period did not differ significantly between both groups. Before weight loss the AO group had slightly greater RMR than the GFO group. After weight loss mean RMR decreased about 10% in the AO group and about 2.5% in the GFO group. Before weight loss DIT was slightly but not significantly higher in the AO group than in the GFO group. After weight loss DIT increased significantly in the GFO group. Weight loss was generally associated with decreased blood glucose, serum triglycerides, and total serum cholesterol levels in the AO women but not in the GFO women.

Body fat distribution and the prognosis for weight reduction: preliminary observations.

In a preliminary study the influence of body fat distribution on the degree of weight reduction, blood lipids and blood glucose was investigated in 17 premenopausal obese women (BMI greater than 27 kg/m2), who followed an energy-reduced diet of 4.2 MJ/day for 8 weeks. Body fat distribution was distinguished in an abdominal and gluteal-femoral type using a cut-off point of 0.80 for the ratio of waist-to-hips girth. Mean weight reduction was about 10 kg. Body fat distribution was not related to the ability to lose weight. Body weight reduction was 10.2 +/- 3.3 kg (mean +/- s.d.) in the abdominal obese (n = 8) and 9.6 +/- 2.4 kg in the gluteal-femoral obese women (n = 8). In abdominal obese women, body fat distribution became more intermediate. This change in body fat distribution coincided in the abdominal obese, after weight loss, with greater decreases in blood glucose and serum lipids than in the gluteal-femoral obese.