Urinary p cresol in autism spectrum diso

  Urinary p-cresol in autism spectrum disorder Antonio M. Persico ⁎ , Valerio Napolioni Child and Adolescent NeuroPsychiatry Unit, University "Campus Bio-Medico", Rome, Italy Dept. of Experimental Neurosciences, IRCCS “Fondazione Santa Lucia”, Rome, Italy a b s t r a c t a r t i c l e i n f o

  ing heritability estimates, and the incomplete penetrance of mutations and/or CNVs often inherited from either parent or even de novo strongly suggest that ASD pathogenesis may be changing over time. The majority of ASD patients are most compatible with a “multiple hit” model, encompassing gene–gene and gene–environment interactions, as well as epigenetic contributions related to several factors, such as increasing parental age at the time of conception ( Leblond et al., 2010; Persico, 2013 ).

  

Neurotoxicology and Teratology

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  E-mail address: a.persico@unicampus.it (A.M. Persico).

  Portillo 21, I-00128 Rome, Italy. Tel.: +39 06 225419155; fax: +39 06 501703333.

  

Neurotoxicology and Teratology 36 (2012) 82–90

⁎ Corresponding author at: Child and Adolescent Neuropsychiatry Unit, Lab. of Mo- lecular Psychiatry & Neurogenetics, University “Campus Bio-Medico”, Via Alvaro del

  In order to determine whether exposure to a given environmental toxicant may contribute to autism pathogenesis in a sizable subgroup of patients, it is important to consider its timing and functional consequences relative to developmental processes. Abnormalities in neurodevelopment must seemingly start during the I–II trimester of prenatal life to yield the spectrum of behavioral abnormalities later diag- nosed as ASD ( ). Hence early prenatal exposure should represent our primary concern. This is well exemplified by at least three evidence-based gene–environment

   ). In summary, increasing prevalence rates, decreas-

  Article history: Received 13 June 2012 Received in revised form 4 September 2012 Accepted 4 September 2012 Available online 10 September 2012 Keywords: Autism Clostridium Gene–environment interactions Gut flora p-cresol p-cresylsulfate

  shared environmental factors has risen to explain as much as 55% of phenotypic variance in a recent twin study Furthermore, only in rare cases is autism fully explained by de novo high-penetrance mutations or by chromosomal rearrangements affecting genes such as NLGN3/4, SHANK3, NRXN1, and MECP2. Recent whole-exome sequencing studies have instead detected a highly heterogeneous collection of de novo mutations distributed in many autism-related genes, collectively increasing disease risk by 5- to 20-fold but nonetheless incompletely penetrant, meaning they are not sufficient to cause the disease

   ) have now dropped down to 37%, while the relative weight of

  ) and 6–10/1000 for broad ASD ( ). Hence this once rare disease has now become one of the most fre- quent conditions in child neuropsychiatry. Genetics strongly contrib- utes to autism pathogenesis. However, heritability estimates above 90% in the early nineties (

  

  1. Introduction Autism spectrum disorder (ASD) is a neuropsychiatric disorder with onset during early childhood and with life-long consequences in the vast majority of cases. It is characterized by impairment in so- cial interaction and communication, as well as by restricted patterns of interest and stereotyped behaviors. Clinical signs and symptom display great interindividual differences in pattern, severity, develop- mental trajectory and treatment response. ASD incidence has dra- matically risen during the last two decades from 2–5/10,000 to approximately 1–2/1000 children for strict autism (

  © 2012 Elsevier Inc. All rights reserved.

  Autism spectrum disorder (ASD) is a neuropsychiatric disorder with onset during early childhood and life- long consequences in most cases. It is characterized by impairment in social interaction and communication, as well as by restricted patterns of interest and stereotyped behaviors. The etiology of autism is highly het- erogeneous, encompassing a large range of genetic and environmental factors. Several lines of evidence sug- gest that, in addition to broader diagnostic criteria and increased awareness, also a real increase in incidence primarily due to greater gene–environment interactions may also be occurring. Environmental exposure to the organic aromatic compound p-cresol (4-methylphenol) is relatively common and occurs through the skin, as well as the gastrointestinal and respiratory systems. However, the largest and most widespread source of this compound is represented by some gut bacteria which express p-cresol synthesizing enzymes not found in human cells. Urinary p-cresol and its conjugated derivative p-cresylsulfate have been found el- evated in an initial sample and recently in a replica sample of autistic children below 8 years of age, where it is associated with female sex, greater clinical severity regardless of sex, and history of behavioral regression. Potential sources of p-cresol excess in ASD, such as gut infection, chronic constipation, antibiotics, abnormal intestinal permeability, and environmental exposure, are being investigated. P-cresol may contribute to worsen autism severity and gut dysfunction, often present in autistic children. It may also contribute to a multibiomarker diagnostic panel useful in small autistic children.

  j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / n e u t e r a interaction models, recently summarized elsewhere (

  Bal-Price et al., 2012 ): (a) RELN and PON1 gene variants +prenatal expo-

  sure to organophosphate pesticides and insecticides; (b) MET gene variants+prenatal exposure to polycyclic aromatic hydrocarbons; and (c) SLC25A12 and ATP2B2 gene variants +prenatal exposure to polychlorinated biphenyls. Secondly, ASD should not be viewed strictly as a “brain disease”: the pathophysiological abnormalities underlying autism indeed are not limited to the central nervous system (CNS), but often involve the immune system and the digestive tract. Many autistic patients display altered T helper 1/T helper 2 ratio, abnormal cytokine profiles, reduced number of lymphocytes and reduced T cell mitogen re- sponse autoimmune disorders cluster in many families with autistic probands ( ); leukocyte gene expression is abnormal, especially for natural killer (NK) cell- related transcripts immune genes are the most overexpressed in post-mortem brains of autistic individuals ( ); anti-brain autoantibodies detected in many autistic children and their mothers can produce behavioral abnor- malities in several animal models, including primates ( ). Also gastrointestinal issues are often noticed by parents in their autistic children, including: (a) constipation; (b) diarrhea; (c) abdominal bloating, discomfort, or irritability; (d) gastro-esophageal reflux or vomiting; (e) feeding issues or food selectivity In- terestingly, dysregulated innate immune defenses have recently been linked with gastrointestinal issues in a subgroup of autistic children who may be especially vulnerable to common microbial dysbiosis ( ).

  83 A.M. Persico, V. Napolioni / Neurotoxicology and Teratology 36 (2012) 82–90

  Fig. 1. Routes of entry for p-cresol into the human body. Red arrows represent environmen- tal exposure through [1] ingestion of food and beverages, [2] skin contact, and [3] inhalation, followed by p-cresol absorption through the upper digestive tract, skin and lungs, respec- tively. Blue arrows represent p-cresol synthesized by gut bacteria from tyrosine or toluene mainly in the colon.

  1.94 Water solubility (25 °C) 21.5 g/l Dissociation constant pKa

  Melting point 35.5 °C Boiling point (1 atm) 201.9 °C Density (20 °C) 1.0178 g/cm ³ Vapor pressure (25 °C) 0.147 mbar Log K ^ow (exp.)

  Table 1 Physico-chemical properties of p-cresol ( OECD, 2003 ).

  from toluene by expressing toluene monooxygenase ( Fig. 3 ). The former pathway is predicted to be significantly more important than the latter, given the much greater availability of L-tyrosine compared to toluene as a substrate in the gut lumen and the broader distribution of p-cresol producing clostridial strains. In fact, exposure to toluene primarily occurs through breathing and skin contact, whereas oral ingestion can occur only through food or water contaminated with this oil derivative.

   ) and Pseudomonas mendocina ( ) produce p-cresol

  Another important source of p-cresol exposure in humans is repre- sented by some gut bacteria, able to express synthetic enzymes not pres- ent in human cells ( Fig. 1 , blue arrows). Two distinct synthetic pathways have been elucidated in gut bacteria: (a) Clostridium difficile, an anaerobic bacterium involved in the most severe forms of antibiotic-associated di- arrhea occasionally leading to pseudomembranous colitis and even to death, expresses p-hydroxyphenylacetate (p-HPA) decarboxylase, able to push the fermentation of tyrosine up to the formation of p-cresol (

  in aerobiosis, while anaerobic degradation plays a marginal role ( OECD, 2003 ).

  p-cresol exposure are listed in Table 2 . P-cresol is readily biodegradable

  , red arrows). The main natural and artificial sources of

   ) ( Fig. 1

  Cresols (o-, m-, p-cresol) are produced in nature through the photo-oxidation of toluene in the atmosphere. Environmental exposure to p-cresol through inhalation, skin contact, ingestion of food and bever- ages is relatively common ( Mandel, 1971; DeBruin, 1976;

  mental behavior and ecotoxicity are its octanol–water partition coeffi- cient (log K ow =1.94), high vapor pressure, water solubility, and pKa value of 10.26, indicating that at environmentally relevant pH values ranging between 5 and 9, p-cresol is largely non-dissociated in aqueous solution.

  Table 1 . Particularly important for environ-

  omatic compounds, which also includes ortho-cresol, meta-phenol, and other mixed formulations. The main physico-chemical properties of p–cresol are reported in

  2.1. Physico-chemical properties P-cresol (4-methylphenol) belongs to the cresol class of organic ar-

  2. P-cresol: general toxicology and implications for human health Information about the general toxicology of p-cresol, including its physico-chemical properties, environmental distribution, use and ex- posure, metabolism, and implications for human health, is available through the Screening Information Data Set (SIDS) of the Organiza- tion for Economic Co-operation and Development (OECD), published by the United Nations Environment Program ( OECD, 2003 ).

2.2. Environmental exposure

10.26 K ow : octanol–water partition coefficient.

84 A.M. Persico, V. Napolioni / Neurotoxicology and Teratology 36 (2012) 82–90

• rainwater
• plants
• petroleum
• tar
• products of volcanic activity
• urines (produced from tyrosine by some gut bac- teria in many mammals, including humans)

• disinfectants and preservatives
• stabilizers in washing and cleaning products
• paints
• fillers
• solvents
• adhesives for surface treatment
• corrosion inhibitors
• impregnation materials
• perfumes and cosmetics
• combustion from incinerators and cigarette smoke
• butylhydroxytoluene and other antioxidants employed as preservatives in foods or as supple- ments
• 4-anisaldehyde (a vanillin-like compound used in the flavor industry)
• intermediates employed in the manufacture of pharmaceutical products
• protective agents for plants
• dyes and pigments
• tomatoes, ketchup
• asparagus
• cheese, butter
• bacon and smoked foods
• red wine
• roasted coffee
• black tea

  Fig. 2. Synthesis of p-cresol from tyrosine by gut bacteria expressing pHPA decarboxylase (hyphenated arrow).

  Foods containing p-cresol:

  Chemical synthetic processes employing p-cresol:

  Artificial sources of exposure:

  Natural sources of exposure:

  p-cresol, leading to the formation of two reactive intermediates, Table 2 Natural and artificial sources of p-cresol exposure.

  disease (see Section 5 below). Using cellular, animal and human pro- tocols, many damaging effects of chronically elevated p-cresol in humans were described, but for at least some of them it is not entirely clear whether and to what extent free p-cresol or p-cresylsulfate are responsible. These effects include:

  p-cresylsulfate that causes many signs and symptoms of chronic renal

  shown that it is instead the accumulation of its conjugated derivative

  P-cresol was initially identified as a uremic toxin, but it was later

   ). Also skin contact can be potentially fatal, causing

  skin corrosion and discoloration, gastrointestinal bleeding, and toxic effects on the nervous system, liver and kidneys ).

  Voluntary or accidental intoxication with p-cresol in humans re- sults in irritation of mouth and throat, abdominal pain, vomiting, he- molytic anemia, cardiovascular disturbances, renal and liver damage, headache, facial paralysis, seizures, coma and death (

  2.4. Acute and chronic toxicity in humans

  Signs of acute toxicity in animals typically include hypoactivity, sal- ivation, tremors and convulsions. The lethal dose 50 (LD50) of oral undiluted p-cresol in rats is 207 mg/kg ( ). Clinical signs of toxicity following inhalation include irritation of mucous mem- branes, excitation and convulsions, hematuria at high p-cresol concen- trations, and death. P-cresol is corrosive to the skin and can cause serious eye damage by contact: the LD50 for dermal application of undiluted p-cresol is 300 mg/kg in rabbits. The “No Observed Adverse Effect Levels” (NOAEL) for p-cresol in mice and rats are generally at or above 50 mg/kg/day, depending on oral, respiratory or dermal absorp- tion ). Fertility is not compromised by p-cresol, which can instead cause fetotoxicity in the form of delayed ossification and re- duced body weight when administered at toxic levels to pregnant rats, but not to rabbits. In vitro, p-cresol does not cause gene mutations in bacterial cells and in mammalian cell systems.

   ).

  in a 95:4:1 ratio. The pivotal role of gut bacteria in synthesizing these urinary compounds is underscored by their significant increase with fasting or slow intestinal transit ( ), and conversely by their decrease with fiber-rich diets or probiotics (

  p-cresylsulfate, p-cresylglucuronide and free p-cresol, approximately

2.3. Metabolism and toxicity in animals

  Free plasma p-cresol and its conjugation derivatives p-cresylsulfate and p-cresylglucuronide are filtered by the renal glomeruli and can be found in the urines of all individuals in small amounts, primarily origi- nating from gut bacteria ( ). It is thus important to underscore that total urinary p-cresol, whose mea- sure is reported in most studies, indeed represents the sum of

  p-cresylglucuronide through glucuronidation, which takes place only in the liver ( Mandel, 1971; DeBruin, 1976; ).

  As partially lipophilic compound, p-cresol travels in the blood most- ly protein-bound ( ) and hypoalbuminemia can indeed increase free p-cresol plasma levels ( ). Only 0.5%–1% of total plasma p-cresol is in free form; approximately 95% of total plasma p-cresol is metabolized to p-cresylsulfate through O-sulfonation, which occurs primarily in colonic epithelial cells and also in the liver; the remaining 3%–4% is metabolized to

• – hepatotoxicity, likely due to inhibition of mitochondrial respira- tion. This would result from the cytochorme P450-mediated oxidation of either the methyl group or the benzene ring of

  

A.M. Persico, V. Napolioni / Neurotoxicology and Teratology 36 (2012) 82–90

  85 Fig. 3. Synthesis of p-cresol from toluene by gut bacteria expressing toluene monooxygenase (hyphenated arrow).

  a quinone methide and a ortho-benzoquinone, respectively, able Interestingly, 12 unaffected siblings of ASD probands displayed to produce toxic effects by alkylating cellular proteins and nucleic intermediate levels. Several members of the C. histolyticum acids ( ); group are known toxin-producers which could both contribute

• – increased endothelial permeability and cardiovascular disease to gut dysfunction and exert systemic effects.

  ( ); found lower levels of Bifidobacteria and

• – immunosuppression and increased susceptibility to infections, higher levels of Lactobacillus species in 58 ASD children com- caused by blunted production of reactive oxygen species by pared to 39 controls. Children with autism had significantly granulocytes, diminished release of IL-12 by immunostimulated lower levels of total short chain fatty acids. Gastrointestinal macrophages, and hampered adhesion of monocytes to endotheli- symptoms were positively correlated with autism severity.

  al cells ( The sensitivity of the microbiological bacterial cultivation

   );

  methods employed in this study is lower compared to the ap-

  Chang

• – inhibition of arachidonic acid-induced platelet aggregation ( proaches applied in the studies summarized above and this

  et al., 2011 );

  methodological discrepancy has likely influenced the consis- growth retardation in weanling pigs ( );

• – tency of the results.

  increased susceptibility to hearing seizures in mice ( – [4] Similarly reduced levels of Bifidobacteria were found by Wang

   );

• – et al. (2011a,b) in faecal samples obtained from 23 individ-

  membrane depolarization through blockade of the delayed-rectifier

  RCK1 (Kv1.1) potassium channel ), which is uals with ASD compared to 9 controls, while 22 unaffected widely expressed in the CNS ( ); siblings displayed intermediate levels. A significant increased lipid peroxidation in rat brain (

• – three-fold reduction in the mucin-degrading bacterium

   ); Akkermansia muciniphila was recorded both in autistic indi-

  decreased Na -K ATPase activity in rat brain (

• – viduals and in their unaffected siblings. Interestingly, the

   ); p-cresol producer Clostridium difficile displayed a dramatic

  inhibition of the conversion of dopamine to noradrenaline (

• – ten-fold mean elevation in ASD patients only (and not in

   );

  their unaffected siblings), with prominent interindividual reduced acetaminophen sulfation capacity, due to competition of

• – variability preventing this result from reaching statistical sig- p-cresol for sulfate conjugation ).

  nificance. [5] Williams et al. (2011) performed ileal biopsies on 15 autistic and 7

  Many of the effects described using cellular and animal models, control children both with gastrointestinal symptoms. Bioptic tis- such as neuronal depolarization, clearly underlie clinically-relevant signs and symptoms of acute intoxication or chronic overexposure sue from ASD children displayed significantly reduced mRNA levels encoding for disaccharidases and hexose transporters. In to p-cresol in humans, including some potentially relevant to ASD. addition, pyrosequencing analysis revealed reduced Bacteroidetes and relatively increased titres of Firmicutes, especially Clostridia.

3. Autism spectrum disorder and the gut flora

  The same group subsequently detected a significant association between the presence of Sutterella species in ileal biopsies and The gut flora is a complex microbial ecosystem that significantly gastrointestinal symptoms in autistic children

   ).

  influences human health ( ). Several studies have assessed the fecal flora of autistic and control in- dividuals, reporting an overgrowth of potentially pathogenic gut mi- [6] Indirect evidence of abnormal gut flora was obtained by

  1 , using a metabolomic approach based on H NMR spec-

  crobial species in a sizable subgroup of autistic patients. Some of troscopy and pattern recognition methods on urine samples of these bacterial strains are known to produce p-cresol:

  Australian autistic and control individuals. Reduced levels of phe- [1] Finegold et al. (2002) initially reported an excess of Ruminococcus nolic compounds including hippurate, phenyacetylglutamine, and Clostridium species, including C. difficile, in fecal samples and p-cresylsulfate characterize the urinary metabolic profile of from 13 ASD patients compared to 8 controls. These Clostridia autistic patients. This result strongly points toward significant groups were then characterized by using a differences in gut microbiomes between patients and controls,

  TaqMan real-time PCR-based approach. Later the same group since the precursors of these three compounds are produced by assessed 33 ASD children, 7 unaffected siblings and 8 controls bacterial metabolism in the gut lumen. The same conclusion is also supported by the higher concentrations of urinary by pyrosequencing, finding Bacteroidetes at high levels in se- verely autistic individuals, whereas Firmicutes were predomi- 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) found nant among controls in ASD children compared to age- and sex-matched controls, as

  [2] Parracho et al (2005) found a higher incidence of the Clostridium well as in an adult with recurrent diarrhea due to C. difficile infec-

  histolyticum group (Clostridium clusters I and II) in the fecal tion ( ). HPHPA derives from dietary phenylalanine

  which, in addition to acting as substrate for phenylalanine

86 A.M. Persico, V. Napolioni / Neurotoxicology and Teratology 36 (2012) 82–90

   ). Finally, in addition to ab-

  Several factors can influence the concentration of endogenous pro- teins in the cecal lumen, including: (a) slow intestinal transit time (i.e., chronic constipation or fasting); (b) greater gut epithelium turnover rates; (c) chronically elevated dietary introduction of tyrosine, taken up into enterocytes and subsequently incorporat- ed into cellular proteins, (d) vastly excessive dietary protein in- take (total proteins or tyrosine-enriched, through milk and its

   ).

  [3] Greater amounts of endogenous proteins in the cecum have been shown to boost p-cresol production, absorption, and urinary ex- cretion rates. Cresol-producing gut bacteria are primarily located in the colon and essentially use tyrosine-containing proteins present in the gut lumen as their substrate. The colon lumen con- tains relatively little polypeptidic material of dietary origin, as this is largely digested and absorbed in the small intestine. In- stead, the colon lumen contains almost entirely endogenous pro- teins, encompassing secreted digestive enzymes, mucus and shed epithelial cells ( ). Importantly, fasting results in elevated serum p-cresol levels positively correlated with cecal protein concentrations in the rat (

  [2] Gut infection with cresol-producing bacterial strains would result in enhanced synthesis of p-cresol from tyrosine in the gut lumen, absorption through the gut wall, and urinary excretion of greater amounts of the compound. An abnormal gut microbiome could represent a spontaneous ASD-related event in some children, while in others it could follow frequent antibiotic treatments. Within this framework, the puzzling age-dependency of urinary p-cresol, elevated both in Italy and in France only among autistic children younger than 8 year of age, could be due to the maturation of the gastroin- testinal immune system and to its increasing ability to control the overgrowth of cresol-producing bacterial strains;

  bring to their mouth objects painted with cresol-containing gloss, “picky eaters” could absorb greater amounts of this com- pound when selecting cresol-enriched foods, while some fam- ilies could be administering cresol-enriched antioxidants to their autistic children, although this practice in relatively un- common in Italy;

  exposure. For example, low-functioning ASD children could

  Several factors could independently contribute to the elevated uri- nary p-cresol levels detected in two ethnically-distinct samples: [1] The excess in p-cresol could largely come from environmental

  4.3. Potential sources of elevated urinary p-cresol in ASD

  4.68%, respectively, of total urinary p-cresol in controls. Statistically signif- icant elevations in ASD children compared to controls were found for uri- nary p-cresylsulfate and p-cresylglucuronate (Pb0.05), whereas free p-cresol displays at this stage a non-significant trend (P=0.086).

  p-cresylglucuronate. These compounds account for 0.06%, 95.26% and

  hydroxylase, is also metabolized by gut bacteria into either phenylpropionic acid or m-tyrosine (3-hydroxyphenylalanine). The latter compound induces catecholamine brain depletion yielding in rats a characteristic behavioral syndrome including forepaw padding, head weaving, backward walking, splayed hind limbs, wet dog shakes, hyperactivity and hyper-reactivity ( ). The studies outlined above generally provide no behavioral descrip- tion of their patient sample, make no attempt to clinically characterize patients with an abnormal gut microbiome and typically disregard the importance of the developmental trajectory of children, which would impose accurate age- and sex-matching between patients and controls. Unfortunately these limitations raise great difficulties in interpreting in- consistencies between different data sets. Nonetheless, collectively these results support the hypothesis that an abnormal gastrointestinal coloni- zation may occur in a subgroup of ASD children. Several factors have been proposed as plausible contributors to this phenomenon, including hypochlorhydria (i.e., absent or low gastric acid production), abnormal gut motility, frequent use of antibiotics, and IgA deficiency. Also inherited or de novo genetic/genomic defects could be involved in some cases. In turn, gut bacteria could exert pathological actions through the direct or indirect production of systemic toxins and neurotoxins, and/or by trig- gering the production of auto-antibodies eventually leading to neuronal damage. To date, several autoantibodies have been found in autistic pa- tients, targeting a variety of endogenous antigens including anti- neuron-axon filament protein, anti-glial fibrillary acidic protein and anti-myelin-basic protein (

  4.2. Preliminary replication findings

  (P b 0.05); (c) children who underwent regression at autism onset, based on parents reporting loss of language skills after acquisition of more than 5 spoken words and loss of social abilities after ini-

  (a) female autistic children compared to males (P b0.05); (b) more severely affected autistic children, regardless of sex

  p-cresol levels were significantly higher among:

  were correlated neither with body mass index nor with urinary cotinine levels, excluding spurious contamination from passive smoking. Instead,

  p-cresol levels normalizing at age 8 and beyond. Levels of p-cresol

  The results summarized in Section 3 , spurred our interest into assessing urinary levels of p-cresol in 59 non-syndromic autistic children and in 59 tightly age- and sex-matched controls Uri- nary p-cresol was measured in first morning urines by high performance liquid chromatography-ultraviolet (HPLC-UV) with multi-wavelength diode array detector (DAD). Urinary concentrations of p-cresol were significantly higher in autistic children compared to controls (123.5± 12.8 vs. 91.2±8.7 μg/ml, Pb0.05). This elevation was surprisingly age-dependent, as it was clearly detectable only up until and including age 7 (134.1±20.1 vs. 70.3±6.7 μg/ml, P=0.005), with urinary

  4. Urinary p-cresol in autism spectrum disorder

  In summary, the relatively high frequency and variable spectrum of gastrointestinal symptoms reported by many parents of autistic children could conceivably stem from a complex combination of abnormal gut microbiome, excessive intestinal per- meability, local immune dysreactivity and possibly pleiotropic roles of autism genes in nervous and gut tissue.

  Magistris et al., 2010 ), though not all studies ( ).

  normal gut colonization, also excessive gut permeability in some autistic children has been reported by several ( D'Eufemia et al., 1996; De

  A replication study is being undertaken on an independent sample of ASD children and controls recruited at the Center for Child and Adolescent Psychiatry of the Hopital Bretonneau in Tours (France). Preliminary anal- yses run on 34 French children already replicate significantly higher uri- nary p-cresol levels in 17 ASD cases compared to 17 matched controls (Pb 0.05). This significant increase occurs only in 8 case-control pairs aged 7 or below (Pb0.01), with no significant difference beyond 7 years of age. Urinary p-cresol is again significantly correlated with clinical sever- ity, as documented using several clinical scales (Antonio Persico, Andrea Urbani, Sonia Cerullo, Catherine Barthelemy, Frederique Bonnet-Briault and Gabriele Tripi, unpublished observation). In addition to measuring total p-cresol, as in our initial study here separately measured its three fractions, namely free p-cresol, p-cresylsulfate and

4.1. Initial findings

  derivatives, soy products, fish, chicken, etc.), and (e) pharmaco- logical inhibition of gastric acid secretion. Interestingly, chronic constipation is one of the most frequent gastrointestinal issues reported by parents with regard to their autistic children ( Buie

  A greater role for p-cresylsulfate rather than for free p-cresol in autism is more compatible with our preliminary re- sults, indicating that the vast majority of total urinary p-cresol excess in ASD children is actually due to p-cresylsulfate. The definition of the functional consequences of excessive p-cresylsulfate blood levels re- quires further investigation, in order to understand whether and to what extent excessive p-cresylsulfate may exert clinically-relevant ef- fects in ASD children, possibly contributing for example to enhanced oxidative stress ( Chauhan and Chauhan, 2006 ).

  p-cresol. For example, p-cresol impairs oxygen-derived free radical

  production by granulocytes in vitro ( ), whereas

  p-cresylsulfate activates free radical production by leukocytes yield-

  ing excessive oxidative stress ( Meert et al.,

  2011 ). Elevated p-cresylsulfate levels in chronic kidney disease have

  been conclusively associated with poor clinical outcome, due to endo- thelial damage and vascular calcifications eventually leading to coro- nary heart disease Wang et

  al., 2010, 2012 ). Similar results were found in diabetic nephropathy

  6. Urinary p-cresol as a potential biomarker for ASD in small children A biomarker can be defined as a biological variable significantly as- sociated with the disease of interest and measurable directly on a given patient or more often on his/her biological specimens/bodily fluids, using sensitive and reliable quantitative procedures. Given the phenotypic heterogeneity of ASD and the well-recognized existence of many “autisms”, each characterized by specific etiopathogenetic under- pinnings ( Persico, 2013

  P-cresylsulfate, now believed to represent the real uremic toxin,

  ), investigators are now striving to define a panel of autism biomarkers able to: (a) foster earlier and more reliable diagnoses, (b) predict developmental trajectories and treatment re- sponse, (c) identify individuals at high-risk, eventually leading to the establishment of preventive health care strategies, (d) contribute to dis- sect ASD into more discrete clinical entities, and (e) possibly even reveal unknown causes or mechanisms of disease. Many autism biomarkers have been proposed to date

  

  ), but scientific, ethical, clini- cal and practical issues still pose a major challenge to their use in clinical practice (

  ). The sensitivity and specificity of each sin- gle biomarker in complex disorders like autism is generally low. The biological complexity of ASD will likely require age- and sex-specific panels, each including several biomarkers belonging to different domains (biochemical, brain imaging, dysmorphological, electrophysi- ological, genetic, immunological, etc). In addition, also some racial and ethnic groups may require specific biomarker panels, not only for predictable specificities in genetic markers conferring autism vulnera- bility, but also for differences in gut-derived compounds. For example,

   report blunted and not increased levels of urinary p-cresylsulfate in autistic patients (see

  tential confounds in clinical features which were not described in this study (autism severity, sex and age distribution, presence of mental re- tardation), using the same technology this same group previously showed dramatic differences in gut flora composition among distinct

  87 A.M. Persico, V. Napolioni / Neurotoxicology and Teratology 36 (2012) 82–90

  interestingly yields functional abnormalities in some cases similar, but in other cases different, or even opposite, to those produced by

  Converging evidence from studies of chronic renal failure begins to sup- port this hypothesis. For many years, p-cresol was regarded as one of the main uremic toxins ): it was believed to accumu- late in the body of patients suffering from chronic renal failure and to produce many signs and symptoms of the disease. It was later realized that a preliminary step in the procedures used to measure p-cresol was the strong acidification necessary for deproteinization; this step resulted in the hydrolysis of p-cresylsulfate and p-cresylglucuronate, spuriously boosting the concentration of free p-cresol ( Lin et al., 2011 ).

  et al., 2010 ). Furthermore, modulating intestinal transit time ei-

   ).

  ther with fasting ( ), or with fiber-rich diets and probiotics (

   ) does result in enhanced and re-

  duced urinary p-cresol levels, respectively, both in rodents and humans; [4] Excessive intestinal permeability (the “leaky gut”), which was pre- viously described in a consistent subgroup of ASD children

  could also fa- cilitate p-cresol absorption. In turn, excessive p-cresol produc- tion and absorption could conceivably alter permeability either directly or through inflammatory mechanisms.

  Studies are ongoing to determine which routes are involved in the enhanced urinary p-cresol excretion we recorded in young autistic chil- dren. Unfortunately urinary p-cresol measures are not correlated with cecal and faecal p-cresol levels, likely due to interindividual variability in liver metabolic rates ). However, while urinary

  p-cresol does not allow immediate inferences about gut-related mech-

  anisms, it does, however, parallel plasma p-cresol levels, which are tox- icologically active over the CNS and other peripheral tissues

  4.4. Other gut-derived bacterial metabolic products possibly relevant to autism P-cresol can essentially originate either from the gut or from environ-

  5. Is p-cresol or p-cresylsulfate the true toxicant? The conjugated derivative p-cresylsulfate represents over 95% of total urinary p-cresol, as measured in our replica sample of French ASD chil- dren (see Section 4.2 ). If pharmacologically active, P-cresylsulfate could thus conceivably represent the true “toxicants”, in addition to or instead of free p-cresol, which by comparison is found only in minute amounts.

  mental exposure ( Fig. 1 ). Interesting parallels can be drawn between

  p-cresol and other behaviorally active, gut-derived bacterial metabolic

  or break-down products. To date, the best-described example possibly relevant to autism is propionic acid (PPA), a short chain fatty acid produced in the gut by anaerobic bacteria including Clostridia and Propionibacteria, through fermentation of dietary carbohydrates and several aminoacids ( ). PPA can also derive from environmental exposure (it is used as a food preservative in many wheat and dairy products) and, differently from p-cresol, it is an endog- enous compound, namely an intermediate of human fatty acid metabo- lism ( ). Intracerebroventricular administration of propionic acid in young rats yields behavioral abnormalities reminiscent of ASD, including perseverance in object-directed behavior, impaired re- versal learning in the T-maze and reduced interaction with a novel rat compared to a novel object ( MacFabe et al., 2011 ). The hippocampus and white matter of these same animals display neuroinflammation in the form of activated microglia and reactive astrogliosis ( MacFabe et

  al., 2011 ), similar to those detected in post-mortem autistic brains

   Also developmental delay and cognitive deficits have been documented in rodents prenatally exposed to PPA ( Brusque

  et al., 1999 ).

  In summary, p-cresol should not be viewed as necessarily unique, but may rather represent one among several gut bacteria-derived compounds able to negatively influence human development and be- havior. Nonetheless, the human clinical data summarized above sug- gest that even in this scenario p-cresol may exert particularly sizable effects, especially in small autistic children.

Section 3 , n.6). In addition to po-

  ( Holmes et al., 2008 ). Conceivably, depending on baseline gut flora composition, similar pathophysiological gut abnormalities in Italian,

  Rapin, 2002 ).

  The authors gratefully acknowledge all the patients and families who participated in our studies, and financial support by the Italian Ministry for University, Scientific Research and Technology, the Italian Ministry of Health, the Fondazione Gaetano e Mafalda Luce (Milan,

  Conflict of interest statement I have no conflict of interests. Acknowledgments

  al abnormalities and cognitive impairment in autistic children. In partic- ular, p-cresol and/or p-cresylsulfate seemingly belong to a restricted set of gut- or environmentally-derived compounds potentially able to worsen behavioral abnormalities and cognitive impairment in small autistic children. Studies performed in specific cellular and animal models, as well as prospective follow-up studies involving baby- siblings (i.e., “high-risk” neonates born to parents with one grown-up child already diagnosed with ASD) will be instrumental in determining whether early prenatal exposure to environment- or maternal gut- derived p-cresol may provide pathogenic contributions, significantly in- creasing the risk of autism spectrum disorder in the offspring. It will also be important to determine the precise origin of elevated p-cresol in small autistic children and to define its influence on the spectrum and intensity of clinical signs and symptoms of ASD, on developmental tra- jectories, and on endophenotypic subgroupings of small children with ASD. Replication studies will also need to determine whether elevated urinary p-cresol/p-cresylsulfate in ASD is specific to some racial and eth- nic groups or represents a generalized finding. If positive, these studies spur hope into the design of cresol-resistant probiotics possibly able to improve behavioral abnormalities when targeted to ASD children with elevated urinary p-cresol.

  p-cresylsulfate as a pathoplastic contributor to the severity of behavior-

  8. Conclusions The currently available evidence summarized in this review pro- vides initial support for postnatal exposure to elevated p-cresol and/or

  animals carrying autism-causing human mutations ( ) should be especially informative in this regard.

   ). Rodent models of autism, such as BTBR mice or transgenic

  at the cellular level how p-cresol and p-cresylsulfate can influence mi- tochondrial function, neurite outgrowth and synaptogenesis are warranted. Moreover, animal models could unveil negative effects on CNS function and development exerted by p-cresol either directly or through its influence on immune parameters and on endothelial permeability (

  p-cresol ). Further studies aimed at assessing

  The behavioral effects of non-toxicological doses of p-cresol in an- imals have not been reported. We are currently performing this study: preliminary data support prominent behavioral effects in BTBR mice following acute i.v. administration of p-cresol doses com- patible with urinary excretion rates recorded in autistic children and in adults with chronic kidney disease (Tiziana Pascucci and Antonio Persico, unpublished data). On the other hand, exploratory cellular studies addressing whether p-cresol and p-cresylsulfate may directly enhance calcium release from intracellular stores or increase calcium entry have yielded negative results (Roberto Piacentini, Claudio Grassi and Antonio Persico, unpublished data), although indi- rect effects could still be mediated by the depolarizing properties of

  In other words, urinary p-cresylsulfate shares conformational fea- tures with MBP so that it cross-reacts with antibodies targeted in progressive multiple sclerosis and molecular mimicry could conceivably trigger autoimmunity also in a subset of autistic chil- dren and of mothers, who produce anti-brain autoantibodies ).

  5. P-cresylsulfate has been identified as the main component of uri- nary myelin basic protein (MBP)-like material ( ).

  4. The membrane depolarizing properties of p-cresol are especially intriguing ), in light of its frequent co-morbidity with hyperactivity and with epilepsy, the latter diag- nosed in as many as 30% of autistic individuals ( Tuchman and

  French and Australian autistics could well yield overgrowth of different bacterial strains, possibly resulting in different urinary patterns of gut-derived compounds.

  impact drug clearance, leading to increased free drug plasma levels and to adverse side effects at the onset of therapy

  p-cresol for both hepatic sulfotransferases and for albumin could

  3. Pharmacological intervention in autistic children could be directly influenced by plasma p-cresol levels. Some autistic children appear exquisitely sensitive to develop side effects to psychoactive drugs at very low doses. , using the analgesic and an- tipyretic drug acetaminophen, found that individuals with high pre-dose urinary levels of p-cresylsulfate had low post-dose uri- nary ratios of acetaminophen sulfate to acetaminophen glucuro- nide. A reduction in liver sulfation capacity, specifically tested using acetaminophen, has also been recorded in low functioning autistic individuals ( ). Thus, competition of

  2. The pro-inflammatory effects of p-cresylsulfate could contribute to enhanced oxidative stress and risk of coronary heart disease in ASD ( Chauhan and Chauhan, 2006; ).

  tion with excessive carbohydrate availability could thus contribute to shape the gut flora in ways yielding gastro- intestinal symptoms in a sizable subgroup of ASD children ( Buie et al., 2010 ).

  p-cresol concentrations in the intestinal lumen, perhaps in conjunc-

  1. P-cresol blocks the growth of many anaerobic bacteria. A few strains, in some cases p-cresol producers but not necessarily, instead tolerate concentrations as high as 0.5% ( ). Elevated

  small children acting through several mechanisms, possibly includ- ing, but not necessarily limited to the following:

  P-cresol and/or p-cresylsulfate could modulate autism severity in

  7. Cellular and systemic actions of p-cresol/p-cresylsulfate potentially relevant to ASD

  among ASD children aged ≤ 7 years, confirming the link between urinary p-cresol and autism severity regardless of sex. It will be interest- ing to verify whether urinary p-cresol holds similar degrees of informa- tiveness in our replication study, once patient recruitment is completed.

  1986 ), were positively correlated with urinary p-cresol concentrations