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Toxicity of formaldehyde and acrolein mixtures: in vitro studies using nasal epithelial cells
ExpToxic Patho11996; 48: 481-483 Gustav Fischer Verlag lena
TNO Toxicology AJ Zeist,The Netherlands
Toxicity offormaldehyde and acrolein mixtures: in vitro studies using nasal epithelial cells* FLEMMING R. CASSEE, WILMA H. STENHUIS, JOHN P. GROTEN and VICTOR J. FERON With I figure and I table Received: December 1,1995; Accepted: December 20,1995 Address for correspondence: Dr. FLEMING R. CASSEE, National Institute of Public Health and the Environment, P.O.box I, 3720 BA Bilthoven, The Netherlands. Key words: Nasal toxicity; Toxicity; Toxicity of formaldehyde, nasal; Formaldehyde toxicity, nasal; Acrolein mixtures, toxicity; Toxicity of acrolein mixtures, nasal; Nasal cytotoxicity; Cytototoxicity, nasal; Nose, cytotoxicity.
Abstract In vitro studies with human and rat nasal epithelial cells were carried out to investigate the combined toxicity of formaldehyde and acrolein and the role of aldehyde dehydrogenases in this process. These studies showed that the toxic effect of mixtures of aldehydes was additive. In addition,aldehyde dehydrogenases wereinhibited by disulfiram and acrolein in S9 incubation but disulfiram did not influence the toxicity in vitro (cell culture). This study does not supportthe idea that aldehyde dehydrogenases playa major role in the detoxification of exogenous aldehydes.
Introduction Formaldehyde (FRM) and acrolein (ACR) are wellknown upper respiratory tract irritants and occur simultaneously as pollutants in many indoor and outdoor environments. The upper respiratory tract and especially the nose is the prime target for inhaled aldehydes. Recently, we have studied possible additive or interactive effects on the nasal epithelium after 3-day inhalation exposure of male Wistar rats to FRM (l.0, 3.2 and 604 ppm), ACR (0.25, 0.67 and lAO ppm) or a mixtures of these aldehydes (CASSEE et al. 1996). As far as nasal microscopic histopathological changes and cell proliferation are concerned neither dose addition nor potentiating interactions occured provided the exposure concentrations were at notoxic-effect levels. The results did not indicate a major role for aldehyde dehydrogenases in the biotransformation of the aldehydes studied. These findings suggest that combined exposure to these aldehydes with the same tar-
* Paper, presented at the 5th International Inhalation Symposium, Hannover, Germany, 20-24 February 1995.
get organ (nose) and exerting the same type of adverse effect (nasal cytotoxicity) is not associated with a greater hazard than that associated with exposure to the individual chemicals, provided the exposure concentrations of each are of no-toxic-effect levels. To study the toxicity at a cellular level we carried out in vitro studies using human and rat nasal epithelial cell lines to assess the combined toxicityof formaldehyde and acrolein and the role of aldehyde dehydrogenases in the (possible) detoxification of exogenous formaldehyde in vitro.
Material and methods Chemicals and cell lines: Formaldehyde (FRM)wasobtained from Janssen Chimica (Beerse, Belgium), acrolein (ACR) from Aldrich Chemie (Brussels, Belgium) and disulfiram (DS) from Sigma (St. Louis, USA). The neutral red dye was obtained from Sigma. The human cell line RPMI2650 (MOORE and SANDBERG 1964) is characterized as an anaplastic squamous cell carcinoma of the nasal septum. The rat cell line FAT2 was kindly provided by Dr. E. BERMUDEZ of the Chemical Industry Institute of Toxicology (Research Triangle Park, NC) and characteristics were recently published (BERMUDEZ et al. 1994). Toxicity assay: Confluent monolayers were incubated for 4 h with mixtures of FRM and ACR or FRM and DS dissolved in Hank's balancedsalt solution. The toxicity was assayed using a neutral red (NR) uptake assay (BABICH and BORENFREUND 1990). After exposure to aldehyde mixtures, cells were incubated with NR for I h. The absorption was measured at 540 nm. Controls were set at 100 % viability. Aldehyde dehydrogenase (ADH) and formaldehyde dehydrogenase (FDH) activitywas assayedaccording to UOTILA and KOIVUSALO (1981) using 1.8mM (highkm) and 45 I-'M (low km) FRM and 4.5 mM glutathione (GSH, Boehringer Mannheim, Germany). The effectsof mixtures were analyzed using the isobolographic method for the 25 % effect. Exp Toxic Pathol48 (1996) 6
481
2000
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observed 25% effect
,
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Additivity envelope -,
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40
60
Fig. 1. An isobologram at the 25 % effect level. The closed circles are the observed effects for combinations of FRM and ACR resulting in a 25 % decrease of the viability.
IJM acrolein
Table 1. Activities of high Km aldehyde dehydrogenase in absence (ADH-) and presence (ADH+) ofGSH and low Km formaldehyde dehydrogenase (FDH; GSH dependent) in S9 homogenate of RPMI2650 and FAT2 cells with (+) and without (-) 40 urn disulfiram (DS).
RPMI2650 - DS +DS FAT2 -DS +DS
ADH - GSH
ADH + GSH FDH
1.76a 0.40 0.55 0.14
1.88 1.74 1.45
1.39
0.66 0.02 0.40
N.D.b
Values are expressed as nmol formaldehyde/min per mg protein; b N.D. =not detectable.
a
Whenever the concentration-effect curves of each of the compounds of the mixture are not linear, the theoretical isobole has to be contructed using mode I and mode II addition. Mode I addition is calculated by taking the increments 482
Exp Toxic Pathol 48 (1996) 6
in the concentration that add up to 25 % effect level starting from zero. In the case of mode II, the effect to be added to the effect EA CR from FRM corresponds to that effect if the cells have been treated with a dose of FRM up to an effect equivalent to EA CR (STEEL and PECKAM 1979). The area enclosed by the mode I and II curves is called the envelope of additivity .
Results Concentration-effect relationships showed a gradual non-linear (loss of the viability) (data not shown). All observed data-points of mixtures of the two aldehydes were within (FAT2) or close to (RPMI2650) the theoretical envelope of additivity. Therefore the effect of combined exposures to FRM and ACR was less than concentration additive for both FAT2 and RPMI2650 cells (fig. 1). FAT2 cells were more sensitive to FRM and ACR compared to RPMI2650 cells (fig. I). Preincubation of cells with DS
(concentrations just below toxicity of OS) did not result in an increased toxicity of formaldehyde. Assaying the aldehyde dehydrogenase activities showed that the activity of AOH without GSH was significantly lower in FAT2 cells than in RPMI2650 cells (table I). The addition of GSHresulted in similaractivities for bothcell lines. The FOH activity (low Km dehydrogenase) in FAT2 was about two thirds of that of the PRMI2650 cells (table I). In addition, it was shown that OS resulted in a decreased ADH activity in the absence of GSH.
Discussion In the present study it was found that the individual concentration-effect curves of the two aldehydes tested were not linear. Therefore one shouldapply mode I and II addition(STEEL and PECKHAM 1979) to create an envelope of additivity. Under this assumption, we conclused that the effects of mixturesof FRM and ACR were additiveor less than additive using dose-addition. The main detoxification pathway of FRM is a conjugation with glutathione and subsequent dehydrogenation by FDH, followed by acetic acid and glutathione formation (SCHAUENSTEIN et al. 1977). ACRis knownto be a veryeffectivein depleting cellular GSH (BEAUCHAMP et al. 1985). Exposureto FRM and ACR wouldbe expectedto result in an increased toxicity of FRM and a more than additive effects might be possible. These hypothesis is not supported by the present results. Furthermore, it was shown that the activities of aldehyde dehydrogenase activities were higher in the RPMI2650 cells, which might explain the difference in toxicity between the two cell lines. However, the inhibition of ADH by disulfiram, (which was shown in using S9 homogenates of the cells), did not result in changes in
the cytotoxicity in vitro. Due to the high reactivity and unspecifity of these aldehydes the adverse action may already have occurred before the aldehydes reach the detoxification enzymes. From these results we concluded that the role of AOH and FOH in the detoxification of FRM in this in vitro system is not as important.
References BABICH H, BORENFREUND E: Applications of the neutral red cytotoxicity assay to in vitro toxicology. ATLA 1990; 18: 857-862. BEAUCHAMP RO, ANDJELKOVICH DA, KLIGERMAN AD, et al.: A critical review of the literature on acrolein toxicity. Crit Rev Toxicol1985; 14: 309-361. BERMUDEZ E, CHEN Z, GROSS EA,et al.:Characterization of cell lines derived from formaldehyde-induced nasal tumors in rats. Molec Carcinogenesis 1994; 9: 193-199. CASSEE FR, GROTEN JP, FERON VJ: Changes in the nasal epithelium of rats exposed by inhalation to mixtures of formaldehyde, acetaldehyde and acrolein. Fund Appl Toxicol1996; 29: 208-219. MOORE GE, SANDBERG AA: Studies of a human tumor cell line withdiploid karyotype. ExpCell Res 1965; 39: 170-175. ROEHM NW, HODGERS GH, HATFIELD SM, et al.: An inproved colorimetric assay for cell proliferation and viability utilizing the tetrazolium saltXTT. J Immul Methods 1991; 142:257-265. SCHAUENSTEIN E, ESTENBAUER H, ZOLLNER H: Aldehydes in biological systems. Pio LTD, London 1977. STEEL GG, PECKHAM MJ: Exploitable mechanisms in combined radiotherapy-chemotherapy: the concept of additivity. 1nt J Radiocation Oncology BioI Phys 1979; 5: 85-91. UOTILA I, KOIVUSALO M: Formaldehyde dehydrogenase. In: JACOBY WB (Ed.): Methods in enzymology 77, Academic Press New York 1981, p. 319.
Exp Toxic PathoI 48 (1996) 6
483
TNO Toxicology AJ Zeist,The Netherlands
Toxicity offormaldehyde and acrolein mixtures: in vitro studies using nasal epithelial cells* FLEMMING R. CASSEE, WILMA H. STENHUIS, JOHN P. GROTEN and VICTOR J. FERON With I figure and I table Received: December 1,1995; Accepted: December 20,1995 Address for correspondence: Dr. FLEMING R. CASSEE, National Institute of Public Health and the Environment, P.O.box I, 3720 BA Bilthoven, The Netherlands. Key words: Nasal toxicity; Toxicity; Toxicity of formaldehyde, nasal; Formaldehyde toxicity, nasal; Acrolein mixtures, toxicity; Toxicity of acrolein mixtures, nasal; Nasal cytotoxicity; Cytototoxicity, nasal; Nose, cytotoxicity.
Abstract In vitro studies with human and rat nasal epithelial cells were carried out to investigate the combined toxicity of formaldehyde and acrolein and the role of aldehyde dehydrogenases in this process. These studies showed that the toxic effect of mixtures of aldehydes was additive. In addition,aldehyde dehydrogenases wereinhibited by disulfiram and acrolein in S9 incubation but disulfiram did not influence the toxicity in vitro (cell culture). This study does not supportthe idea that aldehyde dehydrogenases playa major role in the detoxification of exogenous aldehydes.
Introduction Formaldehyde (FRM) and acrolein (ACR) are wellknown upper respiratory tract irritants and occur simultaneously as pollutants in many indoor and outdoor environments. The upper respiratory tract and especially the nose is the prime target for inhaled aldehydes. Recently, we have studied possible additive or interactive effects on the nasal epithelium after 3-day inhalation exposure of male Wistar rats to FRM (l.0, 3.2 and 604 ppm), ACR (0.25, 0.67 and lAO ppm) or a mixtures of these aldehydes (CASSEE et al. 1996). As far as nasal microscopic histopathological changes and cell proliferation are concerned neither dose addition nor potentiating interactions occured provided the exposure concentrations were at notoxic-effect levels. The results did not indicate a major role for aldehyde dehydrogenases in the biotransformation of the aldehydes studied. These findings suggest that combined exposure to these aldehydes with the same tar-
* Paper, presented at the 5th International Inhalation Symposium, Hannover, Germany, 20-24 February 1995.
get organ (nose) and exerting the same type of adverse effect (nasal cytotoxicity) is not associated with a greater hazard than that associated with exposure to the individual chemicals, provided the exposure concentrations of each are of no-toxic-effect levels. To study the toxicity at a cellular level we carried out in vitro studies using human and rat nasal epithelial cell lines to assess the combined toxicityof formaldehyde and acrolein and the role of aldehyde dehydrogenases in the (possible) detoxification of exogenous formaldehyde in vitro.
Material and methods Chemicals and cell lines: Formaldehyde (FRM)wasobtained from Janssen Chimica (Beerse, Belgium), acrolein (ACR) from Aldrich Chemie (Brussels, Belgium) and disulfiram (DS) from Sigma (St. Louis, USA). The neutral red dye was obtained from Sigma. The human cell line RPMI2650 (MOORE and SANDBERG 1964) is characterized as an anaplastic squamous cell carcinoma of the nasal septum. The rat cell line FAT2 was kindly provided by Dr. E. BERMUDEZ of the Chemical Industry Institute of Toxicology (Research Triangle Park, NC) and characteristics were recently published (BERMUDEZ et al. 1994). Toxicity assay: Confluent monolayers were incubated for 4 h with mixtures of FRM and ACR or FRM and DS dissolved in Hank's balancedsalt solution. The toxicity was assayed using a neutral red (NR) uptake assay (BABICH and BORENFREUND 1990). After exposure to aldehyde mixtures, cells were incubated with NR for I h. The absorption was measured at 540 nm. Controls were set at 100 % viability. Aldehyde dehydrogenase (ADH) and formaldehyde dehydrogenase (FDH) activitywas assayedaccording to UOTILA and KOIVUSALO (1981) using 1.8mM (highkm) and 45 I-'M (low km) FRM and 4.5 mM glutathione (GSH, Boehringer Mannheim, Germany). The effectsof mixtures were analyzed using the isobolographic method for the 25 % effect. Exp Toxic Pathol48 (1996) 6
481
2000
r---------------------,
,:-_. .. ..... ..... ..... .
e
'0
>.
s:
CD '0
.
iii E o
-
1000
---- --
RPMI2650
•
-- --,,
<,
I-
•
observed 25% effect
,
\
\
\
\
\
\
- - mode I . . . mode II
20
CD '0
'=':....• 0. ...... . . . . . . .
>.
CD '0
.. e E
.
• \
40
60
r-------------------------. FAT2
s:
iii
\
;....\.
I
1000
\
500 -
........ ....
••
Additivity envelope -,
'~
.
\
I
\ \ \
\ \
'. \
".1.
20
I
40
60
Fig. 1. An isobologram at the 25 % effect level. The closed circles are the observed effects for combinations of FRM and ACR resulting in a 25 % decrease of the viability.
IJM acrolein
Table 1. Activities of high Km aldehyde dehydrogenase in absence (ADH-) and presence (ADH+) ofGSH and low Km formaldehyde dehydrogenase (FDH; GSH dependent) in S9 homogenate of RPMI2650 and FAT2 cells with (+) and without (-) 40 urn disulfiram (DS).
RPMI2650 - DS +DS FAT2 -DS +DS
ADH - GSH
ADH + GSH FDH
1.76a 0.40 0.55 0.14
1.88 1.74 1.45
1.39
0.66 0.02 0.40
N.D.b
Values are expressed as nmol formaldehyde/min per mg protein; b N.D. =not detectable.
a
Whenever the concentration-effect curves of each of the compounds of the mixture are not linear, the theoretical isobole has to be contructed using mode I and mode II addition. Mode I addition is calculated by taking the increments 482
Exp Toxic Pathol 48 (1996) 6
in the concentration that add up to 25 % effect level starting from zero. In the case of mode II, the effect to be added to the effect EA CR from FRM corresponds to that effect if the cells have been treated with a dose of FRM up to an effect equivalent to EA CR (STEEL and PECKAM 1979). The area enclosed by the mode I and II curves is called the envelope of additivity .
Results Concentration-effect relationships showed a gradual non-linear (loss of the viability) (data not shown). All observed data-points of mixtures of the two aldehydes were within (FAT2) or close to (RPMI2650) the theoretical envelope of additivity. Therefore the effect of combined exposures to FRM and ACR was less than concentration additive for both FAT2 and RPMI2650 cells (fig. 1). FAT2 cells were more sensitive to FRM and ACR compared to RPMI2650 cells (fig. I). Preincubation of cells with DS
(concentrations just below toxicity of OS) did not result in an increased toxicity of formaldehyde. Assaying the aldehyde dehydrogenase activities showed that the activity of AOH without GSH was significantly lower in FAT2 cells than in RPMI2650 cells (table I). The addition of GSHresulted in similaractivities for bothcell lines. The FOH activity (low Km dehydrogenase) in FAT2 was about two thirds of that of the PRMI2650 cells (table I). In addition, it was shown that OS resulted in a decreased ADH activity in the absence of GSH.
Discussion In the present study it was found that the individual concentration-effect curves of the two aldehydes tested were not linear. Therefore one shouldapply mode I and II addition(STEEL and PECKHAM 1979) to create an envelope of additivity. Under this assumption, we conclused that the effects of mixturesof FRM and ACR were additiveor less than additive using dose-addition. The main detoxification pathway of FRM is a conjugation with glutathione and subsequent dehydrogenation by FDH, followed by acetic acid and glutathione formation (SCHAUENSTEIN et al. 1977). ACRis knownto be a veryeffectivein depleting cellular GSH (BEAUCHAMP et al. 1985). Exposureto FRM and ACR wouldbe expectedto result in an increased toxicity of FRM and a more than additive effects might be possible. These hypothesis is not supported by the present results. Furthermore, it was shown that the activities of aldehyde dehydrogenase activities were higher in the RPMI2650 cells, which might explain the difference in toxicity between the two cell lines. However, the inhibition of ADH by disulfiram, (which was shown in using S9 homogenates of the cells), did not result in changes in
the cytotoxicity in vitro. Due to the high reactivity and unspecifity of these aldehydes the adverse action may already have occurred before the aldehydes reach the detoxification enzymes. From these results we concluded that the role of AOH and FOH in the detoxification of FRM in this in vitro system is not as important.
References BABICH H, BORENFREUND E: Applications of the neutral red cytotoxicity assay to in vitro toxicology. ATLA 1990; 18: 857-862. BEAUCHAMP RO, ANDJELKOVICH DA, KLIGERMAN AD, et al.: A critical review of the literature on acrolein toxicity. Crit Rev Toxicol1985; 14: 309-361. BERMUDEZ E, CHEN Z, GROSS EA,et al.:Characterization of cell lines derived from formaldehyde-induced nasal tumors in rats. Molec Carcinogenesis 1994; 9: 193-199. CASSEE FR, GROTEN JP, FERON VJ: Changes in the nasal epithelium of rats exposed by inhalation to mixtures of formaldehyde, acetaldehyde and acrolein. Fund Appl Toxicol1996; 29: 208-219. MOORE GE, SANDBERG AA: Studies of a human tumor cell line withdiploid karyotype. ExpCell Res 1965; 39: 170-175. ROEHM NW, HODGERS GH, HATFIELD SM, et al.: An inproved colorimetric assay for cell proliferation and viability utilizing the tetrazolium saltXTT. J Immul Methods 1991; 142:257-265. SCHAUENSTEIN E, ESTENBAUER H, ZOLLNER H: Aldehydes in biological systems. Pio LTD, London 1977. STEEL GG, PECKHAM MJ: Exploitable mechanisms in combined radiotherapy-chemotherapy: the concept of additivity. 1nt J Radiocation Oncology BioI Phys 1979; 5: 85-91. UOTILA I, KOIVUSALO M: Formaldehyde dehydrogenase. In: JACOBY WB (Ed.): Methods in enzymology 77, Academic Press New York 1981, p. 319.
Exp Toxic PathoI 48 (1996) 6
483