Mercapturic Acids Derived from 2- and 3-Nitrobenzanthrone

Igor Linhart

Department of Organic Chemistry, ICT Prague

Jaroslav Mráz, Iveta Hanzlíková and Emil Frantík

National Institute of public Health, Prague

O

NO2

O

NO2

O

NO2

Nitrobenzanthrones

2-NBA 3-NBA

• Extremely mutagenic pollutants from Diesel emissions, adsorbed on particulate matter.• 3-NBA is one of the strongest mutagens known; “Diesel devil”• First report by Enya, Suzuki et al., 1997• Carcinogenicity confirmed for rats after instillation into trachea (squamous cell carcinoma in lungs)

Sources and exposures

Main source: Combustion proces• Absorbed on soot and other particular matter in diesel and gasoline emissions.• Smoke and soot from burning wood• Atmosferic reaction of benzanthrone with NO2 and O3

3-NBA in diesel exhaust particles (Enya et al. 1997)load of engine rpm concn (μg/g)0% (idling) 600 <0.0016% 1670 0.64780% 1696 6.61

Sources and exposures

Airborne concentrations (Arlt et al., 2005, review)

5.2 – 11.5 pg/m3 Tokyo, Japan

0 – 68.4 pg/m3 semi-rural area in Riso, Denmark

0 – 80 pg/m3 salt mine, Germany

0.4 pg/m3 industrial area, Concord, CA, U.S.A.

Soil concentrations

1.2 – 1158 pg/g Japan

Biological monitoring

Determination of 3-NBA metabolite, 3-aminobenzanthrone (3-ABA) in urine by GC/MS (Grimmer et al., 2000)

Positive findings in salt mine workers (n = 18).

Excretion ranged form 1 – 143 ng/24 h3-ABA

NH2

O

Mutagenicity in Salmonella typhymurium TA98

Compound revertants/nmol1-NBA 5,5602-NBA 3,0003-NBA 208,4009-NBA 84,87011-NBA 61,8-DNP 283,000

Data from Enya, Suzuki et al. 1997; Reynisson et al., 2008

Mutagenicity in S. typhymurium TA98

0

50000

100000

150000

200000

250000

300000

1-NBA 2-NBA 3-NBA 9-NBA 11-NBA 1,8-DNP

Compound

reve

rtant

s/nm

ol

NO2

NO2

1,8-DNP, the most potent mutagen known

O

NO2

O

NHOH

O

NH

OAc

O

NH

OSO3

O

NH

O

NH2

NQO

3-NBA3-OH-ABA

-

OAT

SULT

3-AcO-ABA

3-OS-ABA

nitrenium ion

DNA adducts3-ABA

+

Biotransformation of 3-NBA

Hitherto known aminoarylmercapturic acids

Found also in the urine of rats dosed with aniline or naphthylamine (Boyland et al. 1962 and 1963) but no other aminoarylmercapturic acid s reported thereafter.

NH2 OH NH NH2

S

AcNH COOH

NH2 OH

SNHAc

COOH

NH2

SNHAc

COOH

NH2

NH

NH2 OH

NHNH

+

- H2O+ NAC

2-aminomaphthylmercapturic acid (2-ANMA)

NAC+

aminophenylmercapturic acids

2-APMA 4-APMA

+

+ +

++

- H2O

Papers on mercapuric acids

0

1000

2000

3000

4000

5000

6000

7000

1895-1950 1951-1960 1961-1970 1971-1980 1981-1990 1991-2000 2001-2010 2011-2013

year

No.

of p

aper

s

Publications on mercapturic acids

The absence of reports on aminoarylmercapturic acids since 1963 cannot be explained by a lack of interest in mercapturic acids in general.

According to a SciFinder search in the CAPLUS database

Compound Time (min) Conversion(%)

Product rationOH-ABA : ABA

2-NBA 10 72 80 : 20

2-NBA 25 98 70 : 30

3-NBA 10 89 95 : 5

3-NBA 15 97 80 : 20

Chemical reduction of NBA‘s

O

NHOH

O

NHOH

O

NO2

O

NO2

2-OH-ABA

3-OH-ABA

H2/5% Pd-C

2-NBA

3-NBA

r.t. diglym

H2/5% Pd-C

r.t. diglym

O

NHOH

O

NHOAc

O

NH2

S

AcNH COOH

O

NHOH

O

NHOAc

O

NH2

S

NHAc

COOH

3-OH-ABA

CH3COCN

TBD-methyl polystyrene

NAC

H2O, pH 7.6

3-AcO-ABA 3-ABA-MA

2-OH-ABA

CH3COCN

TBD-methyl polystyrene

NAC

H2O, pH 7.6

2-AcO-ABA 2-ABA-MA

O

NH

O

NH+

+

O

NH

O

NH+

+

Formation of mercapturic acids from 2- and 3-N-hydroxyaminobenzanthrones

Nitrenium ions and their resonance

Determination of ABA-MA in rat urine (HPLC-ESI-MSMS)

ip dosing with 2 mg/kg 2-NBA and 3-NBA

2-NBA

3-NBA

O

NO2

O

NHOH

O

NH2

O

NH2

S

AcNH COOH

O

NHAc

O

NH

OAc

O

NH

OSO3

O

NH

reduction

NQO

reduction

3-NBA 3-OH-ABA 3-ABA

NAT

3-AcABA

-

NATSULT

+

3-AcO-ABA 3-OS-ABA

nitrenium ion

- AcO - SO4--

1. GSH/GST2. M.A.P

3-ABA-MA

Metabolism of 3-NBA in rat in vivo

O

NO2

O

NH2

O

NHAc

reduction NAT

2-NBA 2-ABA 2-AcABA

Metabolism of 2-NBA in rat in vivo

Urinary excretion [% of dose; mean ± S.E., n = 3]

Compound 2-NBA 3-NBA

Metabolite 2-ABATotal (GC/MS)

2-ABAFree

(LC/MS)

2-AcABA

(LC/MS)

3-ABATotal

(GC/MS)

3-ABAFree

(LC/MS)

3-AcABA

(LC/MS)

3-ABA-MA

(LC/MS)

0 - 24 h 0.32±0.06 0.12±0.05 0.24±0.11 0.83±0.40 0.10±0.06 0.41±0.25 0.49±0.15

24 - 48 h 0.008±0.004 < 0.01a < 0.03a 0.04±0.01 < 0.01a < 0.02a 0.02±0.01

Metabolite excretion

Possible protein adduct formation in analogy with GSH-conjugation

protein

O

NH SH

protein

O

NH

O

NH

O

NH

O

NH2

protein

O

NH S

protein

O

NH2

OH

O

NH2

S

O

NH

O

NH2

SG

O

NH2

OH

O

AcNH

S

+

++

+Hydrolysis

protein adduct

GSH +

+M.A.P.

GS-conjugate ABA-MA

ABA-cys

Conclusions

• Significant differences in the chemical reactivity and metabolism between 2- and 3-NBA were observed.

• An unusual mercpaturic acid (ABA-MA) was identified and determined in urine as a new metabolite of carcinogenic 3-NBA but not of its less mutagenic isomer, 2-NBA.

• ABA-MA is a promising “non-invasive“ biological indicator of exposure to 3-NBA but extremely sensitive analytical methods (immunochemical?) will be needed.

• In close analogy to mercapturic acid formation a reaction of the nitrenium ion derived from 3-NBA may lead to binding to cysteine SH groups in globin and other proteins. Proteine adducts formed may be valuable biomarkers of exposure and effect.

Thanks

Jaroslav Mráz, Iveta Hanzlíková, Emil Frantík National Institute of Public Health, Prague, CZ

Alexandra Šilhánková and Michal HimlInstitute of Chemical Technology, Prague, CZ

Financial support by grants 2B08051 NPVII, Ministry of Education,Youth and Sports and NT13401-4/2012, Ministry of Health

…and for your attention