الفهرس 
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Abstract
Summary and Conclusion
Paraphenylene-diamine (PPD) is a synthetic compound that is widely used as a hair dye, in fur and dark cosmetics and temporary tattoo. PPD produces local as well as systemic toxic effects when applied topically and/or ingested. Ingestion of PPD results in acute poisoning with major systemic problem. It results in multisystem involvement as CNS, CVS, renal and musculoskeletal. Transcutaneous absorption of PPD is rapid and may lead to systemic effects including angioedema, gastrointestinal disturbance, tremors, drowsiness, convulsions, dyspnea, liver atrophy, acute renal failure with oliguria, anuria and dark urine (a characteristic chocolate brown color), cardiac arrest or even death. The current study described the systematic approach for an in-depth investigation of PPD intoxication and metabolism. The present study is an experimental study. This study was performed to:•
To develop and validate a new HPLC-DAD method for detection and quantification of p-Phenylenediamine and its metabolites (mono- acetyl p-phenylenediamine and di -acetyl p-phenylenediamine) in the plasma. • To study the applicability of the developed method for determination of different concentrations of PPD and its metabolites in the plasma and tissue samples (kidney and liver) collected from experimental animals.
• To evaluate the dose dependent toxic effects of acute oral and repeated dermal exposure of PPD on different tissues (kidney, liver and heart) of adult male albino rats.
The study was conducted on eighty (80) adult white male albino rats weighing (170–210 gm). The ethics and husbandry conditions of animal research were considered according to the guide of care and use of laboratory animals approved by the ethical committee of Faculty of Medicine, Sohag University.
Animals were divided into 2 groups:
group (A): Represented the acute toxicity group. It included four subgroups (10 animals each)
group IA (control group) was treated with 2 ml of deionized water alone once orally through a gastric tube. group IIA, group IIIA and group IVA: Rats were treated with (PPD) dissolved in 2 ml of deionized water once orally through gastric tube in doses 8, 16 and 80 mg/kg respectively.
group (B): Represented the subchronic toxicity group. It included four subgroups (10 animals each)
group IB (control group) was painted daily with 3ml of deionized water alone. group IIB, group IIIB and group IVB: Rats were painted daily with PPD dissolved in 3ml of deionized water in doses 1, 2 and 3mg/kg.
The current work shows a simple and quick assay. Which will greatly support physicians in hospitals to diagnose patients with PPD intoxication. In addition to previously described unspecific symptoms, this assay allows for a fast and cost-efficient identification of the toxin itself and/ or its metabolites in plasma samples.
As the simultaneous determination of the toxin and the metabolites can be used to determine pharmacokinetics in combination with the development of a first-line treatment before kidney failure occurs.
A simple and reliable HPLC method is described for the simultaneous determination of PPD and its metabolites. Detection was performed by a diode array detector and two different wavelengths (240 and 260) were used for quantitation. The method involved direct extraction procedure by dichloromethane. It is sufficiently rapid and accurate for routine analyses. The current developed method proved to be accurate, sensitive and sufficiently specific. This will be needed in the future to evaluate pharmacokinetic studies regarding PPD.
The method studied in the present work was successfully applied for the identification and quantitation of PPD and its metabolites in experimental animals.
In group IIA and group IIIA, PPD could not be detected in the plasma but still detectable in the kidney and the liver while its metabolites could be detected in plasma, liver and the kidney. PPD and its metabolites (MAPPD and DAPPD) were detected in all target biological samples in group IV.
Acute toxicity study
• There was a significant statistical increase in the mean values of serum urea in all PPD treated acute groups IIA, IIIA, IVA as compared with control group IA.
• On the other hand there was a significant statistical increase in the mean value of serum creatinine only in group IIA as compared with control group.
• There was a significant statistical increase in the mean values of serum AST, ALT and ALP in all PPD treated acute groups IIA, IIIA, IVA as compared with control group IA.
• There was no significant statistical difference in the mean values of serum albumin and total protein in all PPD treated acute toxicity groups IIA, IIIA, IVA as compared with control group IA.
• There was a significant statistical increase in the mean values of serum CPK in all PPD treated acute groups IIA, IIIA, IVA as compared with control group IA.
• Many histopathological changes were observed in PPD treated acute toxicity study groups in comparison with control group. In the kidneys there were vacuolar degeneration, nuclear pyknosis and cytoplasmic vacuolations, brush border damage, widening of urinary space and congestion in glomerular capillaries. These changes increased with increasing the dose. In the liver there were, dilated congested portal vein, necrosis and degenerative changes in periportal hepatocyte with pyknotic nuclei associated with haemorrahge and hydropic degeneration. In the heart there were degeneration of the cardiac muscles fibers with inflammatory cells infiltration. Congestion of the blood vessels also noticed. All these changes were increased with increasing the dose.
Subchronic toxicity study:
• There was a significant statistical increase in the mean values of serum urea and creatinine in all PPD treated subchronic groups IIB, IIIB, IVB as compared with control group IB.
• There was a significant statistical increase in the mean values of serum AST, ALT and ALP in all PPD treated subchronic toxicity study groups IIB, IIIB, IVB as compared with control group IB.
• In spite of significant statistical decrease in the mean values of serum albumin and total protein in all PPD treated subchronic rat groups IIB, IIIB, IVB as compared with control group IB. There was no significant statistical difference between groups IIB and IIIB, groups IIB and IVB and finally between groups IIIB and IVB both for serum albumin and total protein.
• There was a significant statistical increase in the mean values of serum CPK in all PPD treated subchronic rat groups IIB, IIIB, IVB as compared with control group IB.
• Many histopathological changes were observed in PPD treated subchronic toxicity study groups in comparison with control group. In the kidneys there were, widening of the urinary space, glomerular lobulation and tubular degeneration of both proximal and distal convoluted tubules with luminary casts. The interstitium showed inflammatory cells. This increased when the dose increased. In the liver there were dilated congested central vein and blood sinusoids. There was hydropic degeneration of hepatocytes with pyknotic nuclei. In the heart there were degeneration and necrosis of the cardiac muscles fibers with inflammatory cells infiltration. There are multiple areas of muscle bundles separation with inflammatory cells infiltration plus to areas of RBCs extravasations. All these pathological changes were increased with increasing the dose.
• It was found that PPD has multisystem effects (kidney, liver and heart) both functionally and pathologically which increased with increasing the dose and/ or the duration of exposure. These effects were noticed in both (acute oral and subchronic dermal study groups) but they differ in severity.
Conclusion
• A new validated method for the detection and quantification of PPD and its metabolites in plasma samples is presented. Good linearity, LOQ, accuracy, precision, and recovery were demonstrated.
• The current method was applied on different biological samples (plasma, kidney and liver) and its efficiency was approved.
• The study highlighted the experimental correlation between hair dye administration either orally or topically and multi-organs toxic effects include (kidney, liver and heart) both functionally and pathologically which increased with the increase of the dose and the duration of exposure.
• Finally, it was found that at higher doses of PPD, there was broad deviation from the normal values in biochemical parameters and histopathological features compared to controls and lower doses administered.
Recommendations
• It is important that medical physicians be aware of this devastating poison (PPD), as it is freely available and commonly used.
• Increase physicians’ awareness by clinical manifestations of PPD toxicity and lines of management. As early recognition of the complications and prompt treatment are necessary for successful outcome.
• Rapid and accurate management of toxic cases by increasing the number of specialized poison control centers.
• It is important to increase the public awareness regarding the potential lethality of commercial hair dye through a program of health education.
• Governmental legislations and restriction of buying the commercial hair dye to the public should be considered. And providing safe alternative.
• All cosmetics, including hair dye products that are sold in the retail market must have their ingredients listed on the label. Strict measures, certification and licensing should be ensured.
• The public should be aware of the alternate chemical names of PPD such as PARA, 1,4phenylenediamine, 6PPD, p-aminoaniline, phenylenediamine, p-benzenediamine, p-diaminobenzene. 1,4-diaminobenzene, oxidation base 10.
• Increase awareness of hair dressers, workers with rubber, fur, photography and chronic user of hair dye for cosmetic purposes regarding the importance of continuous monitoring of liver and kidney functions. It is important to detect any deviation from normal values as early as possible.
• To avoid occupational contact with PPD to wear protective gloves and masks.
• Social, psychological and economic support should be provided to minimize suicide and other violence forms in the community.
• Containers of cosmetics and hair dyes should be kept out of reach of children.
• Further studies must be done to find effective antidotes against PPD as it is an aggressive rapidly killing poison.