sapporo - coresta | homealtria client services l sept 25, 2012 16 2012_ap08_hayes.pdf congress2012 -...
TRANSCRIPT
Impact on N-nitrosonornicotine (NNN) of varying nitrogen rates for Burley TN90 isolines with variable genetic potential for nicotine to nornicotine conversion. A Hayes, M Lusso, W Morris, A Adams, and K Lion
Sapporo
CORESTA
Congress 2012
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Background
N-Nitrosonornicotine (NNN) has been identified as a harmful or potentially harmful tobacco constituent by FDA.
Previous research has demonstrated that significant reductions in nornicotine results in reduced potential for NNN formation.
Reduced NNN potential has been demonstrated in both Burley lamina and experimental Burley cigarettes.
Lamina nitrate concentration is another important contributor to NNN levels in lamina and smoke.
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Objectives
Evaluate TN90 isolines to assess impact of nicotine to nornicotine conversion rates on NNN levels in cured lamina and smoke. Modulate nitrogen field rates to assess relative impact of
lamina nitrate and lamina nornicotine levels on smoke NNN in experimental Burley cigarettes.
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2011 Field Study
Locations: Glade Spring, VA (4 reps), Blackstone, VA (5 reps) Genotypes: TN90 LC – commercial Burley variety TN90 e4 – stable low converter TN90 ULC – transgenic CYP82E RNAi Treatments: 0 lbs N, 200 lbs N, 400 lbs N
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0 lbs N 200 lbs N 400 lbs N
Topping to harvest interval GS – 5 wks B – 4 wks
Topping to harvest interval GS – 7 wks B – 7 wks
Topping to harvest interval GS – 7 wks B – 7 wks
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Nitrate Source Prob > F Location <.0001 Seed ID 0.3684 N Rate <.0001 Location*Seed ID 0.2787 Location*N Rate 0.0009 Seed ID*N Rate 0.9665
Cured Leaf Nitrate Concentration
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0
2000
4000
6000
8000
10000
12000
14000
0 lbs N 200 lbs N 400 lbs N
Blackstone
0
2000
4000
6000
8000
10000
12000
14000
0 lbs N 200 lbs N 400 lbs N
Glade Spring
Nitrate Range across plots – BLOQ – 19869ppm
NO3 (ppm) NO3 (ppm)
A
B
C
A
B B
* Letters indicate significant differences for Nitrate based on Tukey-Kramer HSD (P<.05)
ANOVA
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Cured Leaf Alkaloid Level
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0
2
4
6
8
0 lbs N 200 lbs N 400 lbs N
Blackstone
0
2
4
6
8
0 lbs N 200 lbs N 400 lbs N
Glade Spring
Total Alkaloids Source Prob > F Location <.0001 Seed ID 0.0445 N Rate <.0001 Location*Seed ID 0.4257 Location*N Rate <.0001 Seed ID*N Rate 0.046
Tot Alk (%DW) Tot Alk (%DW)
A B C
A A A
* Letters indicate significant differences for Alkaloids based on Tukey-Kramer HSD (P<.05)
ANOVA
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0.0%
0.5%
1.0%
1.5%
2.0%
2.5%
3.0%
3.5%
4.0%
4.5%
5.0%
0 lbs N 200 lbs N 400 lbs N
TN 90 LC
TN90 e4
TN90 ULC
A
C
Percent Conversion to Nornicotine by Genotype
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% Conversion Source Prob > F Location 0.0505 Seed ID <.0001 N Rate 0.0002 Location*Seed ID 0.1032 Location*N Rate 0.51 Seed ID*N Rate 0.0111
% Conv
A A
B
* Letters indicate significant differences for % Conversion based on Tukey-Kramer HSD (P<.05). The % Conversion = nornicotine / (nicotine+nornicotine)
ANOVA
B
A
B
C
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0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
TN 90 LC TN90 e4 TN90 ULC
Blackstone
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
TN 90 LC TN90 e4 TN90 ULC
Glade Spring
Cured Leaf NNN Level
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NNN (ppm) NNN (ppm)
A
AB B
A A
B
* Letters indicate significant differences for NNN based on Tukey-Kramer HSD (P<.05) of natural log normalized data
ANOVA
ln(NNN) Source Prob > F Location 0.0011 Seed ID <.0001 N Rate 0.0557
Location*Seed ID 0.0731
Location*N Rate <.0001
Seed ID*N Rate 0.0194
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Cured Leaf %NNN relative to total TSNA
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% NNN Source Prob > F Location 0.022 Seed ID <.0001 N Rate 0.0455 Location*Seed ID 0.0542 Location*N Rate 0.0075 Seed ID*N Rate <.0001 TN90 e4
Mean NNK
Mean NAB
Mean NAT
Mean NNN
TN90 ULC
39%
24%
8%
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Summary 1. Observed variable nitrate and conversion rates in plots 2. Lower nornicotine levels resulted in reduced lamina NNN
2011 Replicated Field Study
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Smoke TSNA Evaluation 1. Selected 6 Plots from each location (12 Total) 2. Handmade cigarettes for each plot from leaf position. 3. Smoke evaluation – 3 replicates under ISO conditions (3 cigs per rep)
Selected Plots – Smoke TSNA evaluation
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Reduced Nornicotine Results in Smoke NNN reduction
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0.00
0.10
0.20
0.30
GTN90 LC 2 GTN90 ULC 4
0.263
0.041
Filler Nornicotine
0
5000
10000
15000
GTN90 LC 2 GTN90 ULC 4
11918 13230
Filler Nitrate
% DW
PPM
TN90 LC G2
TN90 ULC G4
TN90 LC G2
TN90 ULC G4
0.00
0.50
1.00
1.50
2.00
2.50
GTN90 LC 2 GTN90 ULC 4
2.14
0.20
Filler NNN
0.0
5.0
10.0
15.0
20.0
25.0
GTN90 LC 2 GTN90 ULC 4
20.0
5.1
Smoke NNN
PPM
ng/mg TPM A
B
TN90 LC G2
TN90 ULC G4
TN90 LC G2
TN90 ULC G4
* Letters indicate significant differences for smoke NNN based on Tukey-Kramer HSD (P<.05)
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Relative Reduction in NNN in Filler and Smoke
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% DW
PPM
PPM
ng/mg TPM
TN90 LC TN90 ULC TN90 LC TN90 ULC
TN90 LC TN90 ULC TN90 LC TN90 ULC
B
A
D
CD CD C
* Letters indicate significant differences for smoke NNN based on Tukey-Kramer HSD (P<.05)
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Smoke TSNA Evaluation 1. For each location, made 50:50 blends of each possible combination 2. Handmade cigarettes for each blend 3. Smoke evaluation – 3 replicates under ISO conditions
Selected Plots – Smoke TSNA evaluation
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Additive Effect of Nornicotine on Smoke NNN in 50:50 Burley Blends
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0.00
0.10
0.20
0.30
GTN90 LC 2 GTN90 ULC 4
Filler Nornicotine % DW
TN90 LC G2
TN90 ULC G4
0
5000
10000
15000
GTN90 LC 2 GTN90 ULC 4
Filler Nitrate PPM
TN90 LC G2
TN90 ULC G4
15
ng/mg TPM A
B
TN90 LC G2
TN90 ULC G4
C
50:50 G2:G4
* Letters indicate significant differences for smoke NNN based on Tukey-Kramer HSD (P<.05)
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Summary 1. Isolines with reduced nornicotine resulted in significantly lower smoke NNN formation 2. Data suggests nitrate level was a contributor to smoke NNN levels. 3. Burley 50:50 blends showed a general additive trend for smoke NNN levels.
Selected Plots – Smoke TSNA evaluation
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Next Steps 1. 2012 Field Study includes TN90 e4,e5,e10 isoline. 2. Cigarette blend evaluation with Experimental Flue-cured Ultra-low nornicotine tobaccos.
Future studies
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QUESTIONS?
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